Device and methods for monitoring the status of at least one cell

ABSTRACT

A device and methods for monitoring status of at least one cell, wherein the cell has a membrane forming a substantially enclosed structure and defining an intracellular space therein. In one embodiment of the present invention, the device includes a first substrate having a first surface and an opposite second surface, a second substrate supported by the first substrate, the second substrate having a first surface, an opposite second surface, a body portion between the first surface and the second surface, a first side surface and an opposite second side surface, wherein the body portion defines a first passage between the first side surface and the second side surface and an opening on the first surface of the second substrate and in fluid communication with the first passage, and sidewalls positioned above the first surface of the second substrate. In one operation mode, when a first medium is introduced into the first passage, the intracellular space of the cell is in fluid communication with the first passage with the first medium, a sensor measures the response of the cell to the first medium.

The present invention was made with Government support under Grant No.N66001-01-C-8064 awarded by the Defense Advanced Research ProjectsAdministration. The United States Government may have certain rights tothis invention pursuant to these grants.

This application is being filed as a PCT international patentapplication in the name of Vanderbilt University, a U.S. institution(applicant for all designations except the U.S.), and John P. Wikswo, aU.S. citizen and resident (applicant for the U.S. designation), on 6Aug. 2002, designating all countries.

Some references, which may include patents, patent applications andvarious publications, are cited and discussed in the description of thisinvention. The citation and/or discussion of such references is providedmerely to clarify the description of the present invention and is not anadmission that any such reference is “prior art” to the inventiondescribed herein. All references cited and discussed in thisspecification are incorporated herein by reference in their entirety andto the same extent as if each reference was individually incorporated byreference.

FIELD OF THE INVENTION

The present invention generally relates to an apparatus and methods forusing biological material to discriminate an agent. More particularly,the present invention relates to an apparatus and methods that utilize amatrix of biological signatures. In one embodiment, the matrix has aplurality of elements and a dimension of N×M, where N is the totalnumber of the plurality of cells and M is the total number of theplurality of measurable quantities. Thus, the matrix has in total N×Melements, where each element represents a biological signature of one ofa plurality of cells in response to an agent, and each biologicalsignature is one of a plurality of measurable quantities. The presentinvention comprises a method that includes the steps of constructingsuch a matrix of biological signatures, exposing at least one of theplurality of cells to an agent, measuring the measurable quantities ofthe at least one of the plurality of cells responsive to the agent,comparing the measured measurable quantities of the at least one of theplurality of cells responsive to the agent with the correspondingbiological signatures of the matrix of biological signatures, andidentifying the agent from the comparison. The measured measurablequantities can be stored for further processing, analyzing,feed-backing, or the like.

The invention also relates to an apparatus for using biological materialto discriminate an agent. In one embodiment, the apparatus includesmeans for constructing a matrix of biological signatures having aplurality of elements, wherein each element represents a biologicalsignature of one of a plurality of cells in response to an agent, eachbiological signature being one of a plurality of measurable quantities,and wherein the matrix has a dimension of N×M, N being the total numberof the plurality of cells and M being the total number of the pluralityof measurable quantities; means for exposing at least one of theplurality of cells to an agent. The apparatus further includes means formeasuring the measurable quantities of the at least one of the pluralityof cells responsive to the agent, means for comparing the measuredmeasurable quantities of the at least one of the plurality of cellsresponsive to the agent with the corresponding biological signatures ofthe matrix of biological signatures, and means for identifying the agentfrom the comparison.

Certain embodiments of the present invention comprise apparatus andmethods for monitoring the status of a cell that is metabolicallyactive, wherein each metabolic activity of the cell is characterized bya characterization time. More particularly, the apparatus and methodscomprise means and the step for measuring at least one metabolicactivity of the cell at a time period shorter than a characterizationtime corresponding to the measured metabolic activity of the cell,respectively.

Certain other embodiments of the present invention comprise devices andmethods for detecting the response of a plurality of cells to at leastone analyte of interest. More particularly, the devices and methodscomprise means and the steps for contacting the plurality of cells witha plurality of analytes of interest and simultaneously detecting theresponse of the plurality of cells to the plurality of analytes ofinterest, respectively.

Certain further embodiments of the present invention comprise devicesand methods for device for monitoring status of at least one cell,wherein the cell has a membrane forming a substantially enclosedstructure and defining an intracellular space therein. Moreparticularly, the devices and methods comprise means and the steps forproviding a medium into the intracellular space of the cell andmeasuring the response of the cell to the medium, respectively.

Certain other embodiments of the present invention comprise devices andmethods for measuring response of at least one cell to a medium, theresponse of at least one cell to a medium being characterized by areaction time. More particularly, a device of the present inventioncomprises a sensor that measures the response of the cell to the mediumat a time period shorter than the reaction time.

Certain additional embodiments of the present invention comprise devicesand methods for discriminating an agent. More particularly, the devicesand methods comprise means and the steps for constructing a decisiontree having a plurality of branches, each branch corresponding to atleast one defined action, wherein each branch comprises a plurality ofsuccessive branches, each successive branch corresponding to at leastone defined action, providing a conditioned environment sensitive to theagent, obtaining data from response of the agent to the conditionedenvironment, extracting features from the obtained data, selecting abranch from the decision tree corresponding to the features, performingon the features at least one defined action corresponding to the branch,producing a classification of the agent, and iteratively repeating anyor all steps until the agent is discriminated, respectively.

Certain further embodiments of the present invention comprise devicesand methods for discriminating an agent. More particularly, the devicesand methods comprise means and the steps for providing a plurality of Lparameters, L being an integer, each parameter being related to thestatus of the agent, fitting the plurality of L parameters into a set ofith order differential equations, i=1, . . . , N, N being an integer,obtaining a plurality of L features corresponding to L parameters,respectively, from the set of ith order differential equations,separating the L features into a plurality of classes with acorresponding confidence level, providing a plurality of L+1 parameters,each parameter being related to the status of the agent, fitting theplurality of L+1 parameters into a set of ith+1 order differentialequations, obtaining a plurality of L+1 features corresponding to L+1parameters, respectively, from the set of ith+1 order differentialequations, separating the L+1 features into a plurality of classes witha corresponding confidence level, and iteratively repeating any or allsteps until a plurality of classes for the agent is separated with adesired corresponding confidence level, respectively.

Certain other embodiments of the present invention comprise devices andmethods for discriminating an agent. More particularly, the devices andmethods comprise means and the steps for providing a broad spectrumassay having a plurality of L cell lines, L being an integer, each cellline being able to respond to the agent, measuring responses of cellline i, i=1, . . . , L, to the agent, separating the responses intoclass m, m=1, . . . , O, O being an integer and the total number ofavailable classes, with a corresponding robustness factor, selectingcell line j, j=1, . . . , L but ≠i, from the knowledge of class m,measuring responses of cell line j, j=1, . . . , L but ≠i, to the agent,defining a set of feature extraction algorithms from the measuredresponse of cell line j, j=1, . . . , L but ≠i, selecting cell line k,k=1, . . . , L but ≠i and ≠j, measuring responses of cell line k, k=1, .. . , L but ≠i and ≠j, to the agent, separating the responses into classn, n=1, . . . , O, O being an integer and the total number of availableclasses, with a corresponding robustness factor, and iterativelyrepeating any or all steps until a class for the agent with a desiredrobustness factor is obtained, respectively.

BACKGROUND OF THE INVENTION

The biological cell may act as a parallel processing, non-linear,multistate, analog computer. This analog computer can occupy a volume ofless than 10⁻¹⁶ m³ and is primarily powered only by sugars, fats, andoxygen. The complexity of these computers is evidenced by the attemptsto model ongoing biochemical processes based on Mycoplasma genitalium, amicrobe with the smallest known gene set of any self-replicatingorganism (http:\\www.e-cell.org). However, even this simplest modelrequires hundreds of variables and reaction rules, and a complete modeleven for a mammalian cell would be much more complex, requiring inexcess of 10⁵ variables and equations.

Because the cell behaves as an analog computer, it can be programmed.Historically, a limited set of interventions has allowed physiologistsand engineers to study living cells and characterize the feedbackcontrol systems that govern cell function. With the advent of geneticengineering, it is now possible to reprogram the genetic machinery of acell, for example to turn a particular gene on or off, or to producelarge quantities of a particular biochemical. However, there has beenlittle efforts and progress for inserting man-made devices into thecontrol system of a single living cell so as to convert the cell into aprogrammable computational engine.

Therefore, among other things, there is a need to merge cellularbiophysics, microcircuits and microfluidics, and information technologyto create, among other things, programmable microsystems that can beused for sensing, feedback, control and analysis of a single cell and/oran array of interconnected and instrumented living cells.

Additionally, current bio-sensors use biological molecules for specificagent detection via specific binding reactions. However, wide-spectrumdetection is expensive, requiring a priori threat knowledge and a largequantity of specific cells. Assays are susceptible to overload frommultiple threats and false detection and from non-pathogenic “spoof”organisms. Furthermore, addressing new threats involves a lengthy,costly design process. In addition, conventional assays lack cellularmachinery to increase sensitivity.

Therefore, among other things, there is also a need to develop newsystems and methods that are capable of providing a completebio-functional signature of a CBW agent, environmental contaminant,unknown drug, or other threats for better, fast, sensitive accurate andefficient detection.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a device for monitoringstatus of at least one cell, wherein the cell has a membrane forming asubstantially enclosed structure and defining an intracellular spacetherein. In one embodiment, the device includes a first substrate havinga first surface and an opposite second surface, a second substratesupported by the first substrate, the second substrate having a firstsurface, an opposite second surface, a body portion between the firstsurface and the second surface, a first side surface and an oppositesecond side surface, wherein the body portion defines a first passagebetween the first side surface and the second side surface and anopening on the first surface of the second substrate and in fluidcommunication with the first passage, and sidewalls positioned above thefirst surface of the second substrate.

The device also includes a third substrate having a first surface and anopposite second surface. The third substrate, the sidewalls and thesecond substrate define a chamber that is in fluid communication with asecond passage defined by portions of the sidewalls and the thirdsubstrate. The device further includes at least one sensor positioned inthe first passage proximate to the opening, wherein the cell ispositioned in the chamber and the intracellular space of the cell is influid communication with the first passage through the opening of thesecond substrate.

The membrane of the cell defines an opening through which theintracellular space of the cell is in fluid communication with the firstpassage through the opening of the second substrate. The device furtherincludes a punching element positioned underneath the opening of thesecond substrate for making the opening defined by the membrane of thecell. The punching element can be a mechanical device such as apressure-based suction device or an electroporation device such as anelectric potential sucking device.

In one operation mode, when a first medium is introduced into the firstpassage, the intracellular space of the cell is in fluid communicationwith the first passage with the first medium, the sensor measures theresponse of the cell to the first medium.

In another operation mode, when a second medium is introduced into thechamber through the second passage, at least part of the membrane of thecell is in contact with the second medium in the chamber, the sensormeasures the response of the cell to the second medium.

In yet another operation mode, when a first medium is introduced intothe first passage and a second medium is introduced into the chamberthrough the second passage, respectively, the intracellular space of thecell is in fluid communication with the first passage with the firstmedium and at least part of the membrane of the cell is in contact withthe second medium in the chamber, the sensor measures the responses ofthe cell to the first medium and the second medium.

The first passage is in fluid communication with a reservoir of amedium. The device further includes a pair of first controls positionedinside the first passage for controlling the flow of a medium throughthe first passage.

The device second passage is in fluid communication with a reservoir ofa medium. The device also includes a second control positioned insidethe second passage for controlling the flow of a medium through thesecond passage.

In another aspect, the present invention relates to a device formonitoring status of a plurality of cells, wherein each cell has amembrane forming a substantially enclosed structure and defining anintracellular space therein. In one embodiment, the device includes afirst substrate having a first surface and an opposite second surface, asecond substrate supported by the first substrate, the second substratehaving a first surface, an opposite second surface, a body portionbetween the first surface and the second surface, a first side surfaceand an opposite second side surface, wherein the body portion defines afirst passage between the first side surface and the second side surfaceand a plurality of openings distributed on and over the first surface,each opening being in fluid communication with the first passage, and athird substrate having a first surface and an opposite second surfaceand spaced apart from the second substrate thereby defining a spacebetween the second surface of the third substrate and the first surfaceof the second substrate.

The device also includes a plurality of sidewalls positioned between thesecond substrate and the third substrate thereby partitioning the spacebetween the second substrate and the third substrate into a plurality ofchambers above the first surface of the second substrate such that onlyone of openings distributed on and over the first surface is locatedbetween the sidewalls of a corresponding chamber, wherein each chamberis in fluid communication with at least one neighboring chamber througha second passage defined by portions of the corresponding sidewalls andthe third substrate. The device further includes a plurality of sensorspositioned in the first passage, each sensor being proximate to acorresponding one of openings distributed on and over the first surfaceof the second substrate. The plurality of sensors can be substantiallythe same. Or, alternatively, at least two of the plurality of sensorscan be different from each other.

Each cell is positioned in a corresponding one of the chambers and theintracellular space of each cell is in fluid communication with thefirst passage through the opening located between the sidewalls of acorresponding chamber. The membrane of each cell defines an openingthrough which the intracellular space of the cell is in fluidcommunication with the first passage through the opening located betweenthe sidewalls of a corresponding chamber. The device further includes aplurality of punching elements, each positioned underneath an openinglocated between the sidewalls of a corresponding chamber for making theopening defined by the membrane of a corresponding cell. Each punchingelement can be a mechanical device such as a pressure-based suctiondevice or an electroporation device such as an electric potentialsucking device. Punching elements can be same or different.

In one operation mode, when a first medium is introduced into someportion of the first passage, the intracellular space of a cell that isin a chamber corresponding to that portion of the first passage is influid communication with the first passage with the first medium, acorresponding sensor measures the response of the cell to the firstmedium.

In another operation mode, when a second medium is introduced into achamber, at least part of the membrane of a corresponding cell in thechamber is in contact with the second medium, a corresponding sensormeasures the response of the cell to the second medium.

In yet another operation mode, when a first medium is introduced intosome portion of the first passage and a second medium is introduced intoa chamber corresponding to that portion of the first passage,respectively, the intracellular space of a corresponding cell in thechamber is in fluid communication with the first passage with the firstmedium and at least part of the membrane of the corresponding cell is incontact with the second medium, a corresponding sensor measures theresponses of the cell to the first medium and the second medium.

The first passage is in fluid communication with a reservoir of amedium. The device further includes a plurality of first controlspositioned inside the first passage for controlling the flow of a mediumthrough the first passage, wherein for each chamber, a correspondingpair of the first controls controls the flow of the medium throughportions of the first passage under a corresponding chamber.

At least one chamber is in fluid communication with a reservoir of amedium through a second passage. The device also includes a plurality ofsecond controls, each positioned inside a corresponding second passagefor controlling the flow of a medium through that second passage.

In a further aspect, the present invention relates to a method formonitoring the status of at least one cell, wherein the cell has amembrane forming a substantially enclosed structure and defining anintracellular space therein. In one embodiment, the method includes thesteps of confining the cell in a chamber, making an opening in themembrane of the cell, providing a first medium into the intracellularspace of the cell through the opening in the membrane, and measuring theresponse of the cell to the first medium. The method further includesthe steps of providing a second medium into the chamber such that atleast part of the membrane of the cell is in contact with the secondmedium and measuring the response of the cell to the second medium.

In yet another aspect, the present invention relates to a device formonitoring the status of at least one cell, wherein the cell has amembrane forming a substantially enclosed structure and defining anintracellular space therein. In one embodiment, the device includesmeans for confining the cell in a chamber, means for making an openingin the membrane of the cell, means for providing a first medium into theintracellular space of the cell through the opening in the membrane, andmeans for measuring the response of the cell to the first medium. Thedevice further includes means for providing a second medium into thechamber such that at least part of the membrane of the cell is incontact with the second medium and means for measuring the response ofthe cell to the second medium.

In another aspect, the present invention relates to a method formonitoring the status of at least one cell, wherein the cell has amembrane forming a substantially enclosed structure and defining anintracellular space therein. In one embodiment, the method includes thesteps of confining the cell in a chamber, making an opening in themembrane of the cell, providing a first medium into the intracellularspace of the cell through the opening in the membrane, providing asecond medium into the chamber such that at least part of the membraneof the cell is in contact with the second medium and measuring theresponse of the cell to the second medium. The method further includesthe step of measuring the response of the cell to the first medium.

In yet another aspect, the present invention relates to a device formonitoring the status of at least one cell, wherein the cell has amembrane forming a substantially enclosed structure and defining anintracellular space therein. In one embodiment, the device includesmeans for confining the cell in a chamber, means for making an openingin the membrane of the cell, means for providing a first medium into theintracellular space of the cell through the opening in the membrane,means for providing a second medium into the chamber such that at leastpart of the membrane of the cell is in contact with the second mediumand means for measuring the response of the cell to the second medium.The device further includes means for measuring the response of the cellto the first medium.

In a further aspect, the present invention relates to a method forcontrolling the physiological status of at least one cell, wherein thecell has a membrane forming a substantially enclosed structure anddefining an intracellular space therein and controls its physiologicalstatus through an internal cellular control mechanism. In oneembodiment, the method includes the step of providing at least onemedium to the cell such that at least part of the membrane of the cellis in contact with the medium to override the internal cellular controlmechanism. The medium may have an agent.

In one operation mode, the method further includes the steps ofconfining the cell in a chamber and making an opening in the membrane ofthe cell. The providing step further comprises the steps of supplying afirst medium into the intracellular space of the cell through theopening in the membrane, and supplying a second medium into the chambersuch that at least part of the membrane of the cell is in contact withthe second medium. The method further includes the steps of measuringthe response of the cell to the second medium, and adjusting thecomposition of the second medium from the response to affect theoverriding of the internal cellular control mechanism. Moreover, themethod further includes the steps of measuring the response of the cellto the first medium, and adjusting the composition of the first mediumfrom the response to affect the overriding of the internal cellularcontrol mechanism.

In another operation mode, the method further includes the steps ofmonitoring the concentration of at least one selected component of themedium and adjusting the composition of the medium from the monitoredconcentration of at least one selected component of the medium to affectthe overriding of the internal cellular control mechanism. Additionally,the method further includes the steps of s measuring the response of thecell to the medium, and adjusting the composition of the medium from theresponse to affect the overriding of the internal cellular controlmechanism.

In yet another aspect, the present invention relates to a device forcontrolling the physiological status of at least one cell, wherein thecell has a membrane forming a substantially enclosed structure anddefining an intracellular space therein and controls its physiologicalstatus through an internal cellular control mechanism. In oneembodiment, the device includes means for providing at least one mediumto the cell such that at least part of the membrane of the cell is incontact with the medium to override the internal cellular controlmechanism.

The device further includes means for confining the cell in a chamberand means for making an opening in the membrane of the cell. In oneembodiment, the providing means includes means for supplying a firstmedium into the intracellular space of the cell through the opening inthe membrane and means for supplying a second medium into the chambersuch that at least part of the membrane of the cell is in contact withthe second medium. The device further includes means for measuring theresponse of the cell to the second medium and means for adjusting thecomposition of the second medium from the response to affect theoverriding of the internal cellular control mechanism. Moreover, thedevice further includes means for measuring the response of the cell tothe first medium and means for adjusting the composition of the firstmedium from the response to affect the overriding of the internalcellular control mechanism.

Additionally, the device includes means for monitoring the concentrationof at least one selected component of the medium and means for adjustingthe composition of the medium from the monitored concentration of atleast one selected component of the medium to affect the overriding ofthe internal cellular control mechanism. The device further includesmeans for measuring the response of the cell to the medium and means foradjusting the composition of the medium from the response to affect theoverriding of the internal cellular control mechanism.

In a further aspect, the present invention relates to a method forcontrolling the physiological status of at least one cell, wherein thecell has a membrane forming a substantially enclosed structure anddefining an intracellular space therein and controls its physiologicalstatus through an internal cellular control mechanism. In oneembodiment, the method includes the steps of providing at least onemedium to the cell such that at least part of the membrane of the cellis in contact with the medium, monitoring at least one selectedcomponent of the medium, and adjusting the composition of the mediumfrom the monitored concentration of at least one selected component ofthe medium to deliver or remove analytes to the intracellular spacethrough the membrane to affect the internal cellular control mechanism.

In another aspect, the present invention relates to a device forcontrolling the physiological status of at least one cell, wherein thecell has a membrane forming a substantially enclosed structure anddefining an intracellular space therein and controls its physiologicalstatus through an internal cellular control mechanism. In oneembodiment, the device includes means for providing at least one mediumto the cell such that at least part of the membrane of the cell is incontact with the medium, means for monitoring at least one selectedcomponent of the medium, means for adjusting the composition of themedium from the monitored concentration of at least one selectedcomponent of the medium to deliver or remove analytes to theintracellular space through the membrane to affect the internal cellularcontrol mechanism.

These and other aspects will become apparent from the followingdescription of the preferred embodiment taken in conjunction with thefollowing drawings, although variations and modifications therein may beaffected without departing from the spirit and scope of the novelconcepts of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically shows a multicellular bio-silicon hybridmicrosystem according to one embodiment of the present invention.

FIGS. 2A-2B show a PicoCalorimeter or a device according to oneembodiment of the present invention: A. side view and B. top view.

FIGS. 3A-3C show a Microbottle or a device according to one embodimentof the present invention: A. side view; B. top view; and C. sectionalview along line A-A in FIG. 3A.

FIGS. 4A-4C show a Microbottle or a device according to anotherembodiment of the present invention: A. side view; B. top view (with lidremoved); and C. sectional view along line A-A in FIG. 4A.

FIGS. 5A-5C show a Microbottle or a device according to yet anotherembodiment of the present invention: A. side view; B. top view; and C.sectional view along line A-A in FIG. 5A.

FIGS. 6A-6D show a Picocalorimeter or a device according to oneembodiment of the present invention: A. side cross-sectional view alongline D-D in FIG. 6C; B. side cross-sectional view along line C-C in FIG.6C; C. cross-sectional view along line A-A in FIGS. 6A and 6B; and D.cross-sectional view along line B-B in FIGS. 6A and 6B.

FIGS. 7A-7C show a physiometer or a device according to one embodimentof the present invention: A. side sectional view; B. cross-sectionalview along line A-A in FIG. 7A; and C. cross-sectional view along lineB-B in FIG. 7B.

FIG. 8 illustrates an integrated bio-silicon-hybrid system designenvironment according to one embodiment of the invention.

FIG. 9 shows a bio-functional signature matrix according to oneembodiment of the present invention.

FIG. 9A schematically shows a bio-functional signature matrix of FIG. 9in another form according to one embodiment of the present invention.

FIG. 10 shows data of parathion (open symbols) and paraoxon (filledsymbols) on metabolic activity of human hepatocyte and neuroblastomacells according to one embodiment of the present invention.

FIGS. 11A-11C schematically show a sensor head for multispectral readoutaccording to one embodiment of the present invention: A. side sectionalview; B. bottom view; and C. perspective view.

FIGS. 12A-12C schematically show a Nanophysiometer or a device accordingto one embodiment of the present invention: A. side cross-sectional viewalong line A-A in FIG. 12B; and B. top view; and C. exploded view ofpart B in FIG. 12A.

FIGS. 13A-13C schematically show a Nanophysiometer or a device accordingto another embodiment of the present invention: A. side view; and B.cross-sectional view along line A-A in FIG. 13A; and C. enlargement viewof part B in FIG. 13B.

FIG. 14 schematically shows an optical setup for fluorescencemeasurements associated with a Nanophysiometer according to oneembodiment of the present invention.

FIG. 15 schematically shows response of optical beacons to a bindingevent as a means to identify the expression of particular mRNA inresponse to toxins and agents according to one embodiment of the presentinvention.

FIG. 16 illustrates an example of cellular pathways that can bemonitored according to one embodiment of the invention.

FIGS. 17A-17B illustrate an example of toxin discrimination bysimultaneous monitoring of multiple metabolic signals following theexposure of cells to some toxins according to one embodiment of theinvention: A. to DNP; and B. to Cyanide.

FIGS. 18A-18B show discrimination of toxins/agents by monitoringcharacteristic temporal response of cellular phenotypes to toxins,according to one embodiment of the present invention: A. for Macrophage;and B. for Hepatocyte.

FIGS. 19A-19B schematically show discrimination by characteristicresponses in a conditioned environment according to one embodiment ofthe present invention: A. no phenobarbital preexposure; and B. withphenobarbital preexposure.

FIG. 20 shows discrimination by characteristic reaction kinetics ofmetabolic pathways according to one embodiment of the present invention.

FIG. 21 shows the effect of soman on an action potential of a neuronaccording to one embodiment of the present invention.

FIG. 22 is a flowchart illustrating a Process to define a differentialdiscrimination process according to one embodiment of the invention.

FIG. 23 illustrates two signal classification algorithms s according toone embodiment of the invention.

FIG. 24 schematically shows a diagnostics path or process according toone embodiment of the present invention.

FIGS. 25A-25B show a Picocalorimeter or a device according to anotherembodiment of the present invention: A. side cross-sectional view alongline A-A in FIG. 25B; and B. tilted view from the bottom.

FIGS. 26A-26B show an iridium oxide pH electrode forming on a platinuminterdigitated microelectrode array according to one embodiment of thepresent invention: A. a photomicrograph of the electrode array withplatinum, iridium oxide, and platinum microstrips on a glass substrate;B. a pH calibration of the sensor.

FIGS. 27A-27B show a Nanophysiometer or a device according to oneembodiment of the present invention: A. side cross-sectional view; andB. cross-sectional view along line A-A in FIG. 27A.

FIGS. 28A-28C show a Nanophysiometer or a device according to anotherembodiment of the present invention: A. top view; and B. exploded ofpart A in FIG. 28A; and C. cross-sectional view along line B-B in FIG.28B.

FIGS. 29A-29C shows a Nanophysiometer or a device according to yetanother embodiment of the present invention: A. top view; and B.exploded of part A in FIG. 29A; and C. cross-sectional view along lineB-B in FIG. 29B.

FIG. 30 shows a Nanophysiometer or a device according to a furtherembodiment of the present invention in a top view.

FIGS. 31A-31E illustrate the utilization of NanoPhysiometerelectrochemical sensors and their temporal response to changes in pH andoxygen according to one embodiment of the present invention: A. theaverage pH as a function of time in a 100 pL well containing a singlecell with no flow; B. same as FIG. 31A, except plotted as a function oflogarithmic time to show that the response is constant until the protonshave time to diffuse from the cell to the electrode; C. the time takingfor the pH to drop by a certain amount; D. the results of the test ofthe Nanophysiometer with a platinum interdigitated array that sensesoxygen; and E. an individually addressable interdigitated microelectrodearray.

DETAILED DESCRIPTION OF THE INVENTION

Various embodiments of the invention are now described in detail.Referring to the drawings, like numbers indicate like parts throughoutthe views. As used in the description herein and throughout the claimsthat follow, the meaning of “a,” “an,” and “the” includes pluralreference unless the context clearly dictates otherwise. Also, as usedin the description herein and throughout the claims that follow, themeaning of “in” includes “in” and “on” unless the context clearlydictates otherwise. Additionally, some terms used in this specificationare more specifically defined below.

Definitions

The terms used in this specification generally have their ordinarymeanings in the art, within the context of the invention, and in thespecific context where each term is used. For example, conventionaltechniques of molecular biology, microbiology and recombinant DNAtechniques may be employed in accordance with the present invention.Such techniques and the meanings of terms associated therewith areexplained fully in the literature. See, for example, Sambrook, Fitsch &Maniatis. Molecular Cloning: A Laboratory Manual, Second Edition (1989)Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (referredto herein as “Sambrook et al., 1989”); DNA Cloning: A PracticalApproach, Volumes I and II (D. N. Glover ed. 1985); OligonucleotideSynthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames& S. J. Higgins, eds. 1984); Animal Cell Culture (R. I. Freshney, ed.1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. E. Perbal, APractical Guide to Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994). See also, PCR Protocols: A Guide to Methods and Applications,Innis et al., eds., Academic Press, Inc., New York (1990); Saiki et al.,Science 1988, 239: 487; and PCR Technology: Principles and Applicationsfor DNA Amplification, H. Erlich, Ed., Stockton Press.

Certain terms that are used to describe the invention are discussedbelow, or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the devices and methods of theinvention and how to make and use them. For convenience, certain termsare highlighted, for example using italics and/or quotation marks. Theuse of highlighting has no influence on the scope and meaning of a term;the scope and meaning of a term is the same, in the same context,whether or not it is highlighted. It will be appreciated that the samething can be said in more than one way. Consequently, alternativelanguage and synonyms may be used for any one or more of the termsdiscussed herein, nor is any special significance to be placed uponwhether or not a term is elaborated or discussed herein. Synonyms forcertain terms are provided. A recital of one or more synonyms does notexclude the use of other synonyms. The use of examples anywhere in thisspecification, including examples of any terms discussed herein, isillustrative only, and in no way limits the scope and meaning of theinvention or of any exemplified term. Likewise, the invention is notlimited to various embodiments given in this specification.

As used herein, “about” or “approximately” shall generally mean within20 percent, preferably within 10 percent, and more preferably within 5percent of a given value or range. Numerical quantities given herein areapproximate, meaning that the term “about” or “approximately” can beinferred if not expressly stated.

The term “agent” is broadly defined as anything that may have an impacton any living system such as a cell. For examples, the agent can be achemical agent. The chemical agent may comprise a toxin. The agent canalso be a biological agent. Moreover, the agent may comprise at leastone unknown component, which may be identified by practicing the presentinvention. Additionally, the agent may comprise at least one knowncomponent, whose interaction with cells or other components of anenvironment may be detected by practicing the present invention. Theagent can also be a physical agent. Other examples of agent includebiological warfare agents, chemical warfare agents, bacterial agents,viral agents, other pathogenic microorganisms, emerging or engineeredthreat agents, acutely toxic industrial chemicals (“TICS”), toxicindustrial materials (“TIMS”) and the like. Examples of chemical agentsthat may be related to practicing the present invention include Mustard(that may be simulated with chloroethyl ethyl sulphide (endothelia cellsin PC)), GB-Sarin (that may be simulated with Disopropylfluorophosphate(DFP)), VX (that may be simulated with Malathion) or the like. Examplesof viral agents (and their simulants) that may be related to practicingthe present invention include MS2, Hepatitus or simulant or attenuatedvirus, Retroviruses alphaviruses or the like. Examples of bacterialagents (and their simulants) that may be related to practicing thepresent invention include Bacillus globigii or Bacillus subtilis asspore formers similar to anthrax, Erwinia herbicola as a simulant forvegetative bacteria (not sporagenic), E. coli or the like. Additionalexamples of agents can also be found in the following exemplary list ofagents:

-   -   Botulinum Toxin (seven immunological types: A, B, C1, C2, D, E,        F, G)    -   Staphylococcus enterotoxin B    -   Saxitoxin    -   Ricin (Ricinus communis)    -   Epsilon toxin of Clostridium perfringens    -   Mycotoxins    -   Aflatoxins that inhibit DNA and RNA synthesis    -   Anatoxin A    -   Microcystins    -   Cholera Toxin    -   Tetrodotoxin    -   Substance P    -   Bacillus anthracis (Anthrax)    -   Yersinia Pestis, (gram-negative coccobacillus causing the        zoonotic infection Plague)    -   Clostridium botulinum    -   Francisella tularensis (a gram-negative, facultative        intracellular bacterium that causes the zoonosis Tularemia)    -   Brucella spp (spp=several different species?)    -   Burkholderia mallei (Glanders)    -   Burkholderia pseudomallei    -   Chlamydia psittaci    -   Shigella dysenteriae    -   Salmonella spp    -   Vibrio cholerae    -   Cryptosporidium parvum    -   Clostridium perfringens    -   Hepatitis C    -   Variola major (smallpox)    -   Filoviruses/Arenaviruses    -   Alphaviruses    -   Cephalomyelitis Viruses    -   Nipah Virus (a new paramyxovirus)    -   Hantavirus    -   Tick-borne hemorrhagic fevers    -   Dengue (Breakbone or Dandy Fever) fever virus    -   Enteric Viruses    -   Hepatcytes and Hepatitis A    -   Lymphocytes    -   Erythrocytes    -   Endothelial cells    -   HL1 (Cardiac)    -   Secretory cell (depolarize and it secretes things) Beta=insulin    -   PC12 neural cells    -   HELA (Helen Lane)    -   HEK293 Human Epithial Kidney cells    -   Coxiella burnetti    -   Ricksettia prowazekii    -   VX, V-gas    -   G-series (GF-cyclohexyl sarin, GD-Soman, GB-Sarin, GA-Tabun)    -   Mustard Agents    -   HN-1-Nitrogen Mustard    -   HN-2-Nitrogen Mustard (N-Oxide Hydrochloride)    -   Sulfar Mustard    -   Adamsite    -   Arsines    -   Lewisite    -   Hydrogen Cyanide    -   Cyanogen Chloride    -   BZ (Benzphetamine)    -   LSD (Lysergic Acid Diethylamide) (enable comment for this)    -   Chlorine    -   Phosgene    -   CN (2-Chloroacetophenone)    -   Fuel & Combustion Products (Jet Fuels)    -   JP-4    -   JP-8    -   TMPP    -   Herbicides/Pesticides    -   Methyl Parathion (an organophosphorus insecticide)    -   Volatile Organic Carbons (VOC)    -   Benzene    -   Toluene (methylbenzene)    -   Xylene    -   Heavy Metals    -   Lead    -   Chromium    -   Mercury    -   Halogens    -   Fluorine    -   Bromine    -   Cyanides    -   Isocyanates    -   cyanides (as CN)    -   Hydrogen Chloride    -   Sulfur Dioxide    -   Oxides of Nitrogen (NOx)    -   Vinyl Chloride    -   Barium Nitrate    -   Hydrazine    -   DBNP-di-tris-butyl-nitrophenol.

The term “toxin” is broadly defined as any agent that may have a harmfuleffect or harmful effects on any living system such as a cell. Examplesof toxins that may be related to practicing the present inventioninclude cyanide, endotoxin, okadaic acid, Phorbol Myristate Acetate(“PMA”), microcystin, Dinitrophenol (“DNP”), Botulinum toxin (a commonthreat agent; inhibit transmitter release, whole cell MB),Staphylococcus enterotoxin B, ricin (inhibits protein synthesis andribosmone, OT), mycotoxins, aflatoxins, cholera toxin (activates Clpump, vesicle MB, NBR), Saxatoxin or tetrodotoxin (Na channel blocker,vesicle MB), Microcystins (hepatocyte metabolism in PC) andorganophosphates. Other examples of toxins may be also discussedsomewhere else in the specification.

Additional examples of toxins can also be found in the market. Forexample, the following is an exemplary list of toxins with theircorresponding product number that are readily available from acommercial source at gotnet.com:

PRODUCT PRODUCT DESCRIPTION NUMBER Adenylate Cyclase Toxin fromBordetella pertussis 188 Alpha Toxin from Staphylococcus aureus 120Anthrax Lethal Factor (LF), Recombinant from Bacillus anthracis 171Anthrax Protective Antigen (PA), Recombinant from Bacillus anthracis 172Anti-Choleragenoid, Goat Antibody for Cholera Toxin B Subunit 703Anti-Exotoxin A, Goat Antibody for Exotoxin A from Pseudomonasaeruginosa 760 Anti-Toxin A, Goat Antibody for Toxin A from Clostridiumdifficile 752 Anti-VACh Transporter Saporin Conjugate 770 Biotin,Cholera Toxin B Subunit Conjugated 112 Bordetella pertussis, AdenylateCyclase Toxin 188 Bordetella pertussis, Filamentous Hemagglutinin 170Bordetella pertussis, Pertussis Toxin, Liquid in Glycerol Buffer 179ABordetella pertussis, Pertussis Toxin, Lyophilized in Buffer 180Bordetella pertussis, Pertussis Toxin, Lyophilized, Salt Free 181Bordetella pertussis, Pertussis Toxin A Protomer 182 Bordetellapertussis, Pertussis Toxin B Oligomer 183 Botulinum Neurotoxin Type Afrom Clostridium botulinum 130A Botulinum Neurotoxin Type A Heavy Chain132 Botulinum Neurotoxin Type A Light Chain 131 Botulinum NeurotoxinType A Toxoid 133 Botulinum Neurotoxin Type B from Clostridium botulinum136A Botulinum Neurotoxin Type B Heavy Chain 138 Botulinum NeurotoxinType B Light Chain 137 Botulinum Neurotoxin Type B Toxoid 139 CholeraToxin, Azide Free 100 Cholera Toxin from Vibrio cholerae 101 CholeraToxin A Subunit 102 Cholera Toxin B Subunit 103 Cholera Toxin B Subunit,Low Salt 104 Cholera Toxin B Subunit Conjugated to FluoresceinIsothiocyanate 106 Cholera Toxin B Subunit Conjugated to HorseradishPeroxidase 105 Cholera Toxin B Subunit Conjugated toTetramethylrhodamine B 107 Isothiocyanate Cholera Toxin B SubunitConjugated to Phycoerythrin 109 Cholera Toxin B Subunit Conjugated toBiotin 112 Cholera Toxin B Subunit, Recombinant 114 Clostridiumbotulinum, Botulinum Neurotoxin Type A 130A Clostridium botulinum,Botulinum Neurotoxin Type A Heavy Chain 132 Clostridium botulinum,Botulinum Neurotoxin Type A Light Chain 131 Clostridium botulinum,Botulinum Neurotoxin Type A Toxoid 133 Clostridium botulinum, BotulinumNeurotoxin Type B 136A Clostridium botulinum, Botulinum Neurotoxin TypeB Heavy Chain 138 Clostridium botulinum, Botulinum Neurotoxin Type BLight Chain 137 Clostridium botulinum, Botulinum Neurotoxin Type BToxoid 139 Clostridium botulinum, Exoenzyme C3 143 Clostridiumdifficile, Anti-Toxin A, Goat Antibody for Toxin A from Clostridiumdifficile 752 Clostridium difficile, Toxin A 152 Clostridium difficile,Toxin A Toxoid 153 Clostridium difficile, Toxin B 155 Clostridiumtetani, Tetanolysin 199 Clostridium tetani, Tetanus Toxin 190Clostridium tetani, Tetanus Toxin C-Fragment 193 Clostridium tetani,Tetanus Toxoid 191 Corynebacterium diphtheriae, Diphtheria Toxin CRMMutant 149 Corynebacterium diphtheriae, Diphtheria Toxin, Unnicked 150Corynebacterium diphtheriae, Diphtheria Toxoid 151 Diphtheria Toxin CRMMutant 149 Diphtheria Toxin, Unnicked, from Corynebacterium diphtheriae150 Diphtheria Toxoid 151 Enterotoxin Type B from Staphylococcus aureus122 Escherichia coli J5 (Rc), Lipopolysaccharide 301 Escherichia coliK12, D31m4, Primarily Diphosphoryl Lipid A 402 Escherichia coli K12,D31m4 (Re), Lipopolysaccharide 302 Escherichia coli K12 strain LCD25,[³H]Lipopolysaccharide 510 Escherichia coli K12 strain LCD25,Lipopolysaccharide 314 Escherichia coli O111:B4, Lipopolysaccharide 201Escherichia coli O55:B5, Lipopolysaccharide 203 Escherichia coli, StableToxin 118 Exoenzyme C3 from Clostridium botulinum 143 Exotoxin A fromPseudomonas aeruginosa 160 Filamentous Hemagglutinin from Bordetellapertussis 170 Fluorescein Isothiocyanate, Cholera Toxin B SubunitConjugated 106 Fluorescein Isothiocyanate, Tetanus Toxin C-FragmentConjugated 196 Horseradish Peroxidase, Cholera Toxin B SubunitConjugated 105 Horseradish Peroxidase, Tetanus Toxin C-FragmentConjugated 195 Lipid A from Escherichia coli K12, D31m4, PrimarilyDiphosphoryl 402 Lipid A from Salmonella minnesota R595, PrimarilyMonophosphoryl 401 [³H]Lipopolysaccharide from Escherichia coli K12strain LCD25 510 Lipopolysaccharide from Escherichia coli J5 (Rc) 301Lipopolysaccharide from Escherichia coli K12, D31m4 (Re) 302Lipopolysaccharide from Escherichia coli K12 strain LCD25 314Lipopolysaccharide from Escherichia coli O111:B4 201 Lipopolysaccharidefrom Escherichia coli O55:B5 203 Lipopolysaccharide from Salmonellaminnesota R595 (Re) 304 Lipopolysaccharide from Salmonella typhimurium225 Lipopolysaccharide, Ultra Pure from Salmonella minnesota R595 (Re)434 Neuraminidase from V. cholerae 600 Pasteurella Multocida Toxin 156Pertussis Toxin, Liquid in Glycerol Buffer from Bordetella pertussis179A Pertussis Toxin, Lyophilized in Buffer 180 Pertussis Toxin,Lyophilized, Salt Free 181 Pertussis Toxin A Protomer 182 PertussisToxin B Oligomer 183 Pseudomonas aeruginosa, Anti-Exotoxin A, GoatAntibody for Exotoxin A from Pseudomonas aeruginosa 760 Pseudomonasaeruginosa, Exotoxin A 160 Recombinant Adenylate Cyclase Toxin fromBordetella pertussis 188 Recombinant Cholera Toxin B Subunit 114Recombinant protective antigen (PA) from Bacillus anthracis 171Recombinant lethal factor (LF) from Bacillus anthracis 172 Salmonellaminnesota R595, Primarily Monophosphoryl, Lipid A 401 Salmonellaminnesota R595 (Re), Lipopolysaccharide 304 Salmonella typhimurium,Lipopolysaccharide 225 Shiga Like Toxin 1 (Verotoxin 1) 163 Shiga LikeToxin 2 (Verotoxin 2) 164 SNAPtide ™ Peptide Substrate for C. botulinum134 Stable Toxin from Escherichia coli 118 Staphylococcus aureus, AlphaToxin 120 Staphylococcus aureus, Enterotoxin Type B 122 Tetanolysin fromClostridium tetani 199 Tetanus Toxin from Clostridium tetani 190 TetanusToxin C-Fragment 193 Tetanus Toxin C-Fragment Conjugated to Fluorescein196 Tetanus Toxin C-Fragment Conjugated to Horseradish Peroxidase 195Tetanus Toxoid from Clostridium tetani 191 Tetramethylrhodamine BIsothiocyanate, Cholera Toxin B Subunit Conjugated 107 Toxin A fromClostridium difficile 152 Toxin A Toxoid from Clostridium difficile 153Toxin B from Clostridium difficile 155 Tritiated Lipopolysaccharide fromEscherichia coli K12 strain LCD25 510 Verotoxin 1 (Shiga Like Toxin 1)163 Verotoxin 2 (Shiga Like Toxin 2) 164 Vibrio cholerae,Anti-Choleragenoid, Goat Antibody for Cholera Toxin B Subunit 703 Vibriocholerae, Cholera Toxin 101 Vibrio cholerae, Cholera Toxin, Azide Free100 Vibrio cholerae, Cholera Toxin A Subunit 102 Vibrio cholerae,Cholera Toxin B Subunit 103 Vibrio cholerae, Cholera Toxin B Subunit,Low Salt Recommended for Tract Tracing 104

It will be appreciated that all these toxins, in addition to othertoxins given in the specification, are given as specific examples oftoxins that may be related to practicing the present invention. Otherknown or unknown toxins can also be related to or used and may bepreferred for certain, particular applications.

The term “molecule” means any distinct or distinguishable structuralunit of matter comprising one or more atoms, and includes for examplepolypeptides and polynucleotides.

“DNA” (deoxyribonucleic acid) means any chain or sequence of thechemical building blocks adenine (A), guanine (G), cytosine (C) andthymine (T), called nucleotide bases, that are linked together on adeoxyribose sugar backbone. DNA can have one strand of nucleotide bases,or two complimentary strands which may form a double helix structure.“RNA” (ribonucleic acid) means any chain or sequence of the chemicalbuilding blocks adenine (A), guanine (G), cytosine (C) and uracil (U),called nucleotide bases, that are linked together on a ribose sugarbackbone. RNA typically has one strand of nucleotide bases.

As used herein, “cell” means any cell or cells, as well as viruses orany other particles having a microscopic size, e.g. a size that issimilar to that of a biological cell, and includes any prokaryotic oreukaryotic cell, e.g., bacteria, fungi, plant and animal cells. Cellsare typically spherical, but can also be elongated, flattened,deformable and asymmetrical, i.e., non-spherical. The size or diameterof a cell typically ranges from about 0.1 to 120 microns, and typicallyis from about 1 to 50 microns. A cell may be living or dead. As usedherein, a cell is generally living unless otherwise indicated. As usedherein, a cell may be charged or uncharged. For example, charged beadsmay be used to facilitate flow or detection, or as a reporter.Biological cells, living or dead, may be charged for example by using asurfactant, such as SDS (sodium dodecyl sulfate). Cell or a plurality ofcells can also comprise cell lines. Example of cell lines include livercell, macrophage cell, neuroblastoma cell, endothelial cell, intestinecell, hybridoma, CHO, fibroblast cell lines, red blood cells,electrically excitable cells, e.g. Cardiac cell, myocytes (AT1 cells),cells grown in co-culture, NG108-15 cells (a widely used neuroblastoma Xglioma hybrid cell line, ATCC# HB-12317), primary neurons, a primarycardiac myocyte isolated from either the ventricles or atria of ananimal neonate, an AT-1 atrial tumor cardiac cell, Liver cells are alsoknown as Hepatocytes, Secretory cell (depolarize and it secretes things)pancreatic beta cells secrete insulin, HELA cells (Helen Lane), HEK293Human Epithial Kidney c, Erythrocytes (primary red blood cells),Lymphocytes and the like. Each cell line may include one or more cells,same or different. For examples, the liver cell comprises at least oneof Human hepatocellular carcinoma (“HEPG2”) cell, CCL-13 cell, and H4IIEcell, the macrophage cells comprises at least one of peripheral bloodmononuclear cells (“PBMC”), and skin fibroblast cells, the neuroblastomacell comprises a U937 cell, the endothelial cell comprises a humanumbilical vein-endothelial cell (“Huv-ec-c”), and the intestine cellcomprises a CCL-6 cell.

A “reporter” is any molecule, or a portion thereof, that is detectable,or measurable, for example, by optical detection. In addition, thereporter associates with a molecule or cell or with a particular markeror characteristic of the molecule or cell, or is itself detectable, topermit identification of the molecule or cell, or the presence orabsence of a characteristic of the molecule or cell. In the case ofmolecules such as polynucleotides such characteristics include size,molecular weight, the presence or absence of particular constituents ormoieties (such as particular nucleotide sequences or restrictionssites). The term “label” can be used interchangeably with “reporter”.The reporter is typically a dye, fluorescent, ultraviolet, orchemiluminescent agent, chromophore, or radio-label, any of which may bedetected with or without some kind of stimulatory event, e.g., fluorescewith or without a reagent. Typical reporters for molecularfingerprinting include without limitation fluorescently-labeled singlenucleotides such as fluorescein-dNTP, rhodamine-dNTP, Cy3-dNTP,Cy5-dNTP, where dNTP represents DATP, dTTP, dUTP or dCTP. The reportercan also be chemically-modified single nucleotides, such as biotin-dNTP.Alternatively, chemicals can be used that react with an attachedfunctional group such as biotin.

A “marker” is a characteristic of a molecule or cell that is detectableor is made detectable by a reporter, or which may be coexpressed with areporter. For molecules, a marker can be particular constituents ormoieties, such as restrictions sites or particular nucleic acidsequences in the case of polynucleotides. The marker may be directly orindirectly associated with the reporter or can itself be a reporter.Thus, a marker is generally a distinguishing feature of a molecule, anda reporter is generally an agent which directly or indirectly identifiesor permits measurement of a marker. These terms may, however, be usedinterchangeably.

A “measurable quantity” is a physical quantity that is measurable by adevice, or obtainable by simulations. For examples, a measurablequantity can comprise a physical quantity related to cellularphysiological activities of a cell exposed to an agent. Because cellularphysiological activities of a cell involve a lot of activities across awide spectrum, the plurality of physical quantities related to theimpact of the agent on the cell physiology of the cell exposed to theagent are numerous such as heat production, oxygen consumption,uncoupling ratio between heat production and oxygen consumption, freeradical synthesis, fraction of oxygen diverted to free radicalsynthesis, reduced nicotinamide adenine dinucleotide phosphate(“NAD(P)H”), acid production, glucose uptake, lactate release,gluconeogenesis, transmembrane potential, intracellular messengers,membrane conductance, transmembrane pump and transporter rates,messenger RNA expression, neurotransmitter secretion, intracellularglycolytic stores, transmembrane action potential amplitude and firingrate, heat-shock protein expression, intracellular calcium, calciumspark rate and the like.

The term “channel” is broadly defined as any ionic pathway that isassociated with cellular physiological activities of a cell. There arevarious types of channels. For examples, a channel can be aVoltage-gated channel, a Ligand-gated channel, Resting K+ channels (thatare inwardly rectifying K, leak channels), Stretch activated channels,Volume-regulated channels and the like. Examples of Voltage-gatedchannel include K, Na, Ca and Cl. Examples of Ligand-gated channelinclude Neurotranmitter (glutamate {NMDA, AMPA, KAINATE}, GABA, ACH(nicotinic), 5HT, glycine, histamine, Cyclic nucleotide-gated (cAMP,cGMP from inside of cell), some K-selective, some non-specific cationchannels, G-protein activated (mostly potassium; pertussistoxin-inhibited), Calcium-activated (K channels activated by voltage andCa) and the like.

A “sensor” is broadly defined as any device that can measure ameasurable quantity. For examples, a sensor can be a thermal detector,an electrical detector, a chemical detector, an optical detector, an iondetector, a biological detector, a radioisotope detector, anelectrochemical detector, a radiation detector, an acoustic detector, amagnetic detector, a capacitive detector, a pressure detector, anultrasonic detector, an infrared detector, a microwave motion detector,a radar detector, an electric eye, an image sensor, any combination ofthem and the like. A variety of sensors can be chosen to practice thepresent invention.

A “controller” is broadly defined as any device that can receive,process and present information. For examples, a controller can be onemicroprocessor, several microprocessors coupled together, a computer,several computers coupled together, and the like.

The term “biosignature” means a marker for a particular signaling ormetabolic pathway affected by an agent.

The term “analyte” means a material that can be consumed or produced bya cell. Examples of analyte of interest include pH, K, oxygen, lactate,glucose, ascorbate, serotonin, dopamine, ammonina, glutamate, purine,calcium, sodium, potassium, NADH, protons, insulin, NO (nitric oxide)and the like.

The term “flow” means any movement of fluid such as a liquid or solidthrough a device or in a method of the invention, and encompasseswithout limitation any fluid stream, and any material moving with,within or against the stream, whether or not the material is carried bythe stream. For example, the movement of molecules or cells through adevice or in a method of the invention, e.g. through channels of amicrofluidic chip of the invention, comprises a flow. This is so,according to the invention, whether or not the molecules or cells arecarried by a stream of fluid also comprising a flow, or whether themolecules or cells are caused to move by some other direct or indirectforce or motivation, and whether or not the nature of any motivatingforce is known or understood. The application of any force may be usedto provide a flow, including without limitation, pressure, capillaryaction, electroosmosis, electrophoresis, dielectrophoresis, opticaltweezers, and combinations thereof, without regard for any particulartheory or mechanism of action, so long as molecules or cells aredirected for detection, measurement or sorting according to theinvention.

A “medium” is a fluid that may contain one or more agents, one or moreanalytes, or any combination of them. A medium can be provided with oneor more analytes to be consumed by one or more cells. A medium can haveone or more analytes generated by one or more cells. A medium can alsohave at the same time one or more analytes to be consumed by one or morecells and one or more analytes generated by one or more cells.

An “inlet region” is an area of a microfabricated chip that receivesmolecules or cells for detection measurement. The inlet region maycontain an inlet channel, a well or reservoir, an opening, and otherfeatures which facilitate the entry of molecules or cells into thedevice. A chip may contain more than one inlet region if desired. Theinlet region is in fluid communication with the main channel and isupstream therefrom.

An “outlet region” is an area of a microfabricated chip that collects ordispenses molecules or cells after detection, measurement. An outletregion is downstream from a discrimination region, and may containbranch channels or outlet channels. A chip may contain more than oneoutlet region if desired.

An “analysis unit” is a microfabricated substrate, e.g., amicrofabricated chip, having at least one inlet region, at least onemain channel, at least one detection region and at least one outletregion. A device of the invention may comprise a plurality of analysisunits.

A “main channel” is a channel of the chip of the invention which permitsthe flow of molecules or cells past a detection region for detection(identification), or measurement. The detection and discriminationregions can be placed or fabricated into the main channel. The mainchannel is typically in fluid communication with an inlet channel orinlet region, which permit the flow of molecules or cells into the mainchannel. The main channel is also typically in fluid communication withan outlet region and optionally with branch channels, each of which mayhave an outlet channel or waste channel. These channels permit the flowof molecules or cells out of the main channel.

A “detection region” or “sensing volume” or “chamber” is a locationwithin the chip, typically in or coincident with the main channel (or aportion thereof) and/or in or coincident with a detection loop, wheremolecules or cells to be identified, characterized, hybridized,measured, analyzed or maintained (etc.), are examined on the basis of apredetermined characteristic. In one embodiment, molecules or cells areexamined one at a time. In other embodiments, molecules, cells orsamples are examined together, for example in groups, in arrays, inrapid, simultaneous or contemporaneous serial or parallel arrangements,or by affinity chromatography.

A “branch channel” is a channel which is in communication with adiscrimination region and a main channel. Typically, a branch channelreceives molecules or cells depending on the molecule or cellcharacteristic of interest as detected by the detection region andsorted at the discrimination region. A branch channel may be incommunication with other channels to permit additional sorting.Alternatively, a branch channel may also have an outlet region and/orterminate with a well or reservoir to allow collection or disposal ofthe molecules or cells.

A “gene” is a sequence of nucleotides which code for a functionalpolypeptide. For the purposes of the invention a gene includes an mRNAsequence which may be found in the cell. For example, measuring geneexpression levels according to the invention may correspond to measuringmRNA levels. “Genomic sequences” are the total set of genes in aorganism. The term “genome” denotes the coding sequences of the totalgenome.

“Preconditioning” is the process by which the physiological state of acell or cells is adjusted by application of a known drug, toxin,analyte, or other chemical or physiological stimulus for the purpose ofadjusting the response of the cell to a subsequently applied toxin. Forexample, if a cell is in a resting state, an agent that decreasesmetabolic level may not alter the cell's metabolism below thealready-low resting state. But if the cell is preconditioned to be in alevel of high metabolic activity, the subsequent application of thatsame agent would produce a much larger signal.

“Feedback” refers to the process by which a measured signal is amplifiedand transformed in a manner that it can be used to control or alter theproperty of the system in a manner that in turn affects the measuredvariable. Negative feedback would be feedback applied in a manner toreduce the amplitude of the measured variable. Positive feedback wouldbe feedback applied in a manner to increase the amplitude of themeasured variable.

“Actuator” is a device that can, under electrical, mechanical, orchemical control, or the like, perform an action in such a manner as toeffect a change to a system. For example, a valve is an actuator thatcan control the release of an analyte.

“Feedback Control” is the process by which sensors and actuators areused to control the state of a system by means of positive or negativefeedback, or both, such that the state of the system either remainsconstant in time or changes in accord with a desired sequence ofchanges. For example, the sensing of intracellular pH could be used toincrease the flow of fluidic media into a cell to wash away the protonsthat are acidifying the sensing volume as a result of cell metabolism.As another example, a glucose sensor that detects a decrease in theglucose level in the sensing volume could use an actuator to increasethe inflow of glucose into the sensing volume to stabilize the glucoselevels to which the cell is exposed despite metabolic changes thataffect the cell's utilization of glucose. The feedback signal can inturn provide direct information about, for example, the glucoseconsumption of the cell.

“Reaction time” is the time that a system of interest requires torespond to a change. For example, the reaction time of a cell is thetime required for at least one of the physiological processes of a cellto adapt or respond to the application of an agent. The reaction time ofa sensor is the time required for the sensor to respond to a change inthe quantity that it is sensing. For example, the reaction time of anelectrochemical sensor is set by the size of the sensor and thethickness and nature of protective coatings on the activated surfaces ofthe sensor. The reaction time of a microfluidic system is determined bythe reaction time of the cell to changes in the environment, the timerequired for chemical species to diffuse throughout the sensing volume,the reaction time of the sensor(s), the reaction time of the actuatorsand the diffusion time of the analyte being controlled by the actuators.It follows that stable feedback control of a physiological parameterrequires that the diffusion, sensor and actuator reaction times are lessthan the reaction time of the cell.

Overview of the Invention

In one aspect, the present invention relates to a system and methods forusing biological material to discriminate an agent. In one embodiment asshown in FIG. 1, a system 100 according to the present inventionincludes a plurality of cells 105, where each cell has multiplemetabolic pathways 104 for metabolic events. The system 100 furtherincludes a first structure 101 for receiving the plurality of cells toform a biolayer, where the first structure 101 has a plurality ofsensing volumes, and each sensing volume is in a conditioned environmentcapable of receiving and maintaining at least one cell. As such formed,cells 105 may be coupled together and communicate to each other.

The system 100 additionally includes an array 102 of sensors 106positioned underneath the biolayer 101 for simultaneously monitoring ofmultiple metabolic pathways 104 for each of the plurality of cells,where each metabolic pathway may be disturbed in the presence of anagent (not shown). The system 100 further includes at least onecontroller 107 coupled to each sensor 106 of the array 102.

When an agent invades the conditioned environment, the array of thesensors 102 detects the changes of metabolic events for at least one ofthe cells and generates at least one signal in response, and thecontroller 107 receives the signal from the array of sensors 102 andidentifies the agent from the signal. The controller 107 furtherincludes means for quantifying the agent from the measured response.Thus, among other things, contrary to traditional approaches todiscriminate an agent from testing the agent, one aspect of the presentinvention is to discriminate, and quantify, an agent from the responseof a living cell to the agent.

Moreover, because a living cell behaves as an analog computer, it can beprogrammed. However, the cell controls its physiological status throughan internal cellular control mechanism. Therefore, in order to programthe cell, i.e. direct the cell to do what it is taught to do, theinternal cellular control mechanism of the cell has to be overridden.Historically, a limited set of interventions has allowed physiologistsand engineers to study living cells and characterize the feedbackcontrol systems that govern cell function. With the advent of geneticengineering, it is now possible to reprogram the genetic machinery of acell, for example to turn a particular gene on or off, or to producelarge quantities of a particular biochemical. However, as yet there hasbeen little work on inserting man-made devices into the control systemof a single living cell so as to convert the cell into a programmablecomputational engine. The present invention merges cellular biophysics,microcircuits and microfluidics, and information technology to createprogrammable Multicellular Bio-Silicon Hybrid Microsystems such assystem 100 as shown in FIG. 1, which serve as biological computingengines having an array of interconnected and instrumented living cellswith associated control and modeling software; and a biophysicalinfospace design environment required to program and analyze output fromthese Microsystems.

Thus, as shown in FIG. 1, in addition to sensors 106, the physical layer102 may further include microbottles, picocalorimeters, microfluidics,and controllers, some of them according to the present invention arediscussed in more detail below. Additionally, the system 100 hasinfolayer 102 that may have reconfigurable digital and analog software,programmable digital signal processors (DSPs) and at least onecontroller 107 (which may itself be a DSP), which provides an integratedcomputational structure to receive measurements and compute signalidentification procedures to detect and identify agents includingtoxins, and control cellular actuators. Sensors 106 can be multispectralsensors that measure and transduce multiple cell parameters and controlcell environmental parameters via actuators and effectors. The system100 may further have a biophysical infospace design environment 108 thatincludes software CAD/CASE tools that allow user(s) 109 to designalgorithms for the computational structure 113 which supports multiplecustomized interfaces for the users 109 who, for examples, may includemicrobiologists, hardware/sensor engineers, diagnostic experts and thelike. The computational structure 113 includes system models such ascellular metabolic processes and modeling 110, physical sensor andeffectors on the sensor system 111, and identification and diagnosisprocedures and decision models 112. Software generators 114, which maybe embedded in one or more computers such as a network, automaticallyconvert models 110, 111, and/or 112 into executable code(s) to programthe infolayer 103 including controller 107, which in turn communicatesand controls with the biolayer 101 through the physical layer 102, andto drive biological simulators 115, whose output 116 can be used toverify algorithms and procedures defined in generators 114 prior toimplementation in the biolayer 101, the physical layer 102, and theinfolayer 103.

Accordingly, the system 100 provides a programmable cellular microsystemthat has a true bi-directional, bioionic-silicon interface. Developmentof the system 100 and related devices involves not only the building ofcell-based biosensors, but also the creation of biological andsolid-state processes needed to form a functioning assembly of sensorsand actuators. One challenge is to identify the computations or tasksfor which this technology is best suited. Nevertheless, the presentinvention provides multiple biosilicon microsystems that can be combinedto form larger analog biomicrocomputers capable of solving particularclasses of problems with higher speed and lower power consumption thancould be implemented in silicon and software.

In one application, the system 100 can be utilized to discriminate anagent. In one embodiment, at least one cell 105 is provided and isexposed to the agent, which may be contained in a medium, the responseof the cell to the agent is measured in terms of a physical quantityrelated to at least one of the cellular physiological activities of thecell, and from the measured response the agent can be identified.Furthermore, the agent (such as its concentration in the medium) can bequantified from the measured response. When cell(s) are used as a canaryto detect an agent, the present invention has the tremendous advantageof non-specificity, in that it reveals information only about overallcellular metabolic activity and hence it is not necessary to develop aparticular sensor for each anticipated agent such as toxin.

In another embodiment, at least one cell is provided and is exposed tothe agent, which may be contained in a medium, the response of the cellto the agent is measured, where the response of the cell to the agent ischaracterized by a reaction time, at a time period shorter than thereaction time, and from the measured response the agent can beidentified. Furthermore, the agent (such as its concentration in themedium) can be quantified from the measured response. The response cantake various forms including a temporal response of the cell to theagent, which is measured in at least two measurements. The time betweenthe measurements is shorter than the reaction time corresponding to thetemporal response of the cell. Indeed, as discussed below, among otherthings, one aspect of the present invention is that it provides devicesand methods in which the diffusion time from the cell to the sensor iscomparable to the response time of the sensor such that the response ofthe cell to the agent can be measured faster and better than what priorart could offer.

Exemplary devices and methods according to the embodiments of thepresent invention are given below. Note that titles or subtitles may beused in the examples for convenience of a reader, which in no way shouldlimit the scope of the invention.

EXAMPLES Example 1 Biosignatures Matrix

In one aspect of the present invention, a wide-spectrum,activity-detection technology is developed that employs several novelcell and membrane-based sensing technologies, in order to provide acomplete bio-functional signature of a CBW agent, unknown drug, or otherthreat. The bio-functional signatures can be used with advancedalgorithms to discriminate between different agents. The system anddevices are extraordinarily versatile and general; because one uniquefeature of the present invention, among other things, is that thebiological impact of the toxins is detected and measured, rather thanthe toxins themselves.

Today, biosensors use biological molecules (antibodies, enzymes, nucleicacids, etc.) for specific agent detection via specific bindingreactions. Wide-spectrum detection is expensive, requiring a priorithreat knowledge and a large quantity of specific cells. Assays aresusceptible to overload from multiple threats and false detection andfrom non-pathogenic ‘spoof’ organisms. Furthermore, addressing newthreats involves a lengthy, costly design process. In addition,conventional assays do not employ cellular machinery to increasesensitivity.

An alternative is to monitor the state of a set of optimized biologicalsystems so that a departure from normal homeostasis sounds an alert of apossible CB attack. A broad set of physiological tests on a combinationof receptor, ion-channel, cell, and tissue-based biosensors can providea rapid, sensitive, and accurate differential diagnosis of cellularpathophysiology. One challenge is to develop sound methods for achievingclear signatures of the patho-physiological effects of CBW agents. Thisapproach discerns both the identities of known CBW agents, and themechanism of action for unknown agents. Such information will help guidecountermeasures.

Accordingly, a spectrum of on-line biosensors for physiologicalresponses in model systems or living cells are developed, in order toobtain and discriminate bio-functional signatures of CBW agents. Thesebiosensors can measure heat generation, metabolic products, ion-channelconductance, transmembrane potential, intracellular conductance, theexpression of optically tagged proteins for cardiac myocytes, neurons,and endothelial cells, and intracellular and intercellular signalling,which includes the secretion of neurotransmitters, hormones, and growthfactors. These modalities are chosen to span the broad range ofphysiological mechanisms affected by the spectrum of possible CBWagents. The multi-phasic measurements can be used to tracktoxin-induced, temporal responses, and test hypotheses regardingprophylactic or therapeutic measures in support of a differentialdiagnosis.

Referring now to FIG. 9, in one embodiment, the present inventionrelates to a method for using biological material to discriminate anagent. A matrix of biological signatures 900 constructed according toone embodiment of the present invention is shown in FIG. 9. Matrix 900conceptually represents a hypothetical table or process used to specifycell species, measurement methods, and expected/measured responses fordefinition of identification algorithms related to the discrimination ofagents. As shown in FIG. 9, column 901 represents cell species, i.e.,cells, which are utilized to discriminate an agent. Column 902represents devices that are used to make corresponding measurements,where each measurement measures a biological signature of one of aplurality of cells in response to an agent. Note that while some ofdevices given in column 902 are examples according to embodiments of thepresent invention, which are disclosed in this specification, otherdevices and even some existing technologies can be utilized to practicethe present invention. Column 903 represents measurable quantities orattribute/product to be measured. Column 904 represents outputs of themeasurements, i.e., the expected or measured response of thecell/attribute for each of the agents of interest, including the changefrom nominal/steady state for a cell and the signal-to-noise of themeasurement. Furthermore, the matrix 900 is an open-ended, i.e., it canbe expanded to include specifications for additional cell types 905 aswell as identification for additional agents 906. In other words, thetotal number of elements for column 902 (number of cells), N, the totalnumber of elements for column 903 (number of measurable quantities) andeven total number of elements for agents 906 (number of agents) areadjustable. It means that, for example, it does not require thedevelopment of specific assays to new agents such as known or unknowntoxin threats. This generality arises because the present inventionallows one to measure the biological impact of toxins rather than thetoxins themselves.

FIG. 9A shows a matrix of biological signatures 950 constructedaccording to another embodiment of the present invention. As shown inFIG. 9A, matrix 950 has a dimension of N×M, where N represents cellspecies or cell lines such as HeLa, NB, and HepG2. Each cell line mayhave a number of cells participating. Thus, N may also reflect the totalnumber of cells that are utilized to discriminate an agent.

M represents the total number of the plurality of measurable quantitiessuch as pH, DO, Glucose, Lac, CO₂, NADH as shown in FIG. 9A. When theone or more cells 951 are exposed to an agent such as a toxin, one ormore measurable quantities 953 of the one or more cells responsive tothe agent are measured, generating a plurality of outputs 957. Each ofthe outputs 957 is an element of the matrix 950 that represents abiological signature corresponding to a particular cell responsive tothe agent. For examples, element 961 represents the measurement ofanalyte pH for an HepG2 cell responsive to toxin 954, element 963represents the measurement of analyte DO for an HepG2 cell responsive totoxin 954, etc. The measured measurable quantities of the plurality ofcells 951 responsive to the toxin 954, i.e., outputs 957, can then becompared with the corresponding biological signatures of the matrix 950,which can be obtained through calibrating the matrix from a plurality ofchemical agents once the matrix is formed and stored in a model database. From the comparison, the toxin can be identified. Note that eachoutput 957 has amplitude, from which the toxin can be quantified throughthe comparison, which is another unique feature of the present inventionover the existing technologies. The measured measurable quantities,outputs 957, can be stored in a database associated with a memory device(not shown) for further processing, analyzing, feedbacking, or the like.

Outputs 957 can be obtained in several ways. In one embodiment, forexample, one can start to measure element 961 in the first row, thenelement 963, until all elements in the first row of the matrix 950 havebeen measured. Then, one can continue to measure element 971 in thesecond row, then element 973, until all elements in the second row ofthe matrix 950 have been measured. This process is repeated for the restrows of the matrix 950 until all biology signatures corresponding to theelements in all N rows of the matrix have been measured. This process ofmeasurements may be termed as an orthogonal measurement. Note that theelements in all N rows of the matrix can be measured simultaneously.Alternatively, the elements in all N rows of the matrix can be measuredin sequence, or any way one chooses to proceed. It will be appreciatedthat the method described above is just one of many ways to get theelements of the matrix measured. For example, one is free to pick anyelement of the matrix as a starting point to measure. Alternatively, onecan pick several (up to all) elements of the matrix to be measuredsimultaneously.

Calibration(s) may be performed before the measurements. Moreover,before the measurements, preconditioning agents may be applied to thecells 951 to place the cells in a desired physiological state. Cells 951can be placed in one or more chambers 958. Each chamber 958 may receiveone or more cells. Additionally, during the measurements, a mediumcontaining analytes may be supplied to cells in each chamber so as tomaintain a preconditioned environment to keep the cells of interestalive. Different chambers may receive different mediums in term ofcontent through proper fluid control. Moreover, the exposure of thecells to the agent needs to be kept under a threshold of exposure forirreversible cell damage or cell death to keep the cells of interestalive. The exposure of the cells to the agent can be adjusted accordingto the measured measurable quantities. Some or all of the activitiesdiscussed above can be coordinated, performed, or processed by acomputer or a computer associated with a network.

Outputs 957 can be obtained through various apparatus. In one embodimentas shown in FIG. 9A, a sensor array 952 is utilized to measure themeasurable quantities of at least one of the plurality of cellsresponsive to the agent. Sensor array 952 includes a plurality ofsensors, which may be same or different. Some or all of them may bedevices provided by the present invention such as sensor 956, which is aNanoPhysiometer as discussed in more detail below. Sensor array 952 maybe considered as a matrix of sensors corresponding to the matrix ofbiological signatures 900.

Referring now to FIG. 10, response of cells to certain toxin is shown.In FIG. 10, measured acidification rate of cultured cells, when exposedto a stepped increase in a toxin, followed by washout. Line 1001represents acidification response of hepatocytes to parathion. Line 1002represents response of hepatocytes to paroxon. Line 1003 representsresponse of neuroblastomas to parathion. And line 1004 representsresponse of neuroblastomas to paroxon. FIG. 10 shows the dose-responseof a change in pH induced by agents in cell cultures of μL volumes. FIG.10 uses published data of parathion (open symbols) and paraoxon (filledsymbols) on metabolic activity of human hepatocyte and neuroblastomacells obtained with a commercially available CytoSensor™ instrument,which also shows that commercially available instruments may be modifiedand utilized to practice the present invention.

FIG. 15 shows the response of optical beacons to a binding event as ameans to identify the expression of particular mRNA in response totoxins and agents. Upon exposure to Interferon-Y at 1501, synthesis ofmRNA is triggered at 1502. The resulting mRNA 1504 then binds with amolecular beacon 1503 in a manner that the ends of the molecular beaconare no longer in close proximity, so that the resulting beacon-mRNAcomplex fluoresces 1505. FIG. 16 schematically indicates that whenhybridized with a complementary oligonucleotide, the hairpin structurelinearizes, distancing the fluorophore and quencher to yieldfluorescence. In other words, existing optical molecular beacontechnology can also be utilized practice the present invention.

An example of cellular pathways can be monitored with discriminationmatrix 950 and sensor array 957 of the present invention. Cellularprocesses are metabolically-driven, energy-requiring events. The basalenergy requirements are derived from the oxidation of metabolicsubstrates, e.g., glucose 1602, either by oxidative phosphoralation 1611involving the aerobic TCA or Kreb's cycle 1609 or anerobic glycolysis1602. When glycolysis is the major source of energy, the metabolicactivity of cells can be estimated by monitoring the rate at which thecells excrete acidic products of metabolism 1605, e.g., lactate 1606 andCO₂ 1607. In the case of aerobic metabolism, the consumption ofextracellular oxygen 1603 and the production of oxidative free radicals1604 are reflective of the energy requirements of the cell.Intracellular oxidation-reduction potential can be measured byautofluorescent measurement of the NADH 1611 and NAD⁺ 1610 ratio. Theamount of energy, e.g., heat 1608, released by the cell is derived fromanalytical values for substances produced and/or consumed duringmetabolism which under normal settings can be predicted from the amountof oxygen consumed (4.82 kcal/l O₂). The coupling between heatproduction and oxygen utilization can be disturbed by toxins. Directmicrocalorimetry measures the temperature rise of a thermally isolatedsample. Thus when combined with measurements of oxygen consumptioncalorimetry can used to detect the uncoupling activity of toxins. Thedevices disclosed in this specification are designed to measure, amongother things, the following variables: glucose 1602, lactate 1606, CO₂1607, NADH 1611 and NAD⁺ 1610 ratio, heat 1608, O₂ consumption 1603, andfree-radical production 1604. Some metabolic activities of cells ofinterest are listed in the following Table 1.1.

TABLE 1.1 Glucose + 2 ADP + 2 NAD⁺ → 2 Pyruvate + 2 ATP + 2 NADHPyruvate + NADH → Lactate + NAD⁺ Pyruvate + CoA + FAD + → 3 CO₂ +FADH2 + GDP + 3 NAD⁺ + NAD(P)⁺ GTP + 3 NADH + NAD(P)H 0.5 O₂ + 3 ADP +NADH → 3 ATP + NAD⁺ 0.5 O₂ + 2 ADP + FADH₂ → 2 ATP + FAD

The energy requiring events within the cells are sensitive to theavailability of energy in the form of ATPase and NADH (NADPH) to sustainthe activity. Those energy-consuming events include maintenance of themembrane potential, intracellular pH, and osmotic balance. Moreover,many of the cell signaling events that control cell growth, programmedcell death (apoptosis), cellular cytoskeleton and cell specific function(e.g., immune response of macrophages and gluconeogenesis and albuminsynthesis by hepatocytes) are very sensitive to metabolic stress. Thus,one aspect of the present invention is to take advantage of the uniquecharacteristics of cells to develop signatures that will allow fordiscrimination. For example, the sodium potassium ATPase, which is themajor consumer of ATPase in the resting cell, is reliant on adequatecellular ATPase availability to maintain a transmembrane potential.Without this potential, cell viability is dramatically compromised.Toxins that target the pump or the cellular ATPase levels will produceidentifiable and measurable signatures.

The approach to monitor specific metabolic pathways has the tremendousadvantage of non-specificity, in that it reveals information aboutoverall cellular metabolic activity and hence it is not necessary todevelop a particular sensor for each anticipated toxin. Yet bymonitoring specific features of the metabolic response in multiple cellstypes, we can define the discrimination algorithm. Clearly the responseto a toxin can be cell specific. For example, the ECBC laboratorydemonstrated that parathion and paraoxon have opposite effects onhepatocyte and neuroblastoma cell metabolism. The cell lines utilized inthe present invention include, for examples, macrophages (PBMC,U937),liver (HEPG2,CCL-13,H4IIE), neural (HTB-11) and endothelial (HUV-ECC-C),and intestinal (CCL-6) cells. They represent cells that are derived fromorgans, which are targets of biotoxins. The liver is a major target oftoxins (aflotoxin, organophosphates, viral hepatitis) both because ofits anatomical location, (i.e., is exposed to all toxins absorbed viathe alimentary tract) and because it is metabolically active and playssuch a central role in biodetoxification in the organism. The intestineis directly exposed to toxins (e.g., bacteria, virus, enterotoxin)entering via the oral route. Neuronal cells are targets of a number oftoxins (organophosphates) that alter ion channel function.

Macrophages serve as one of the most important sentinels for thepresence of many biotoxins. They are ideally located at the major routesof potential toxin entry: respiratory airways, intestine, liver andskin. The alveolar macrophage (AM) lives on the mammalian bronchialsurfaces and is exposed to inhaled polluted air. Acting as a scavenger,it protects the pulmonary tissue from invading microorganisms andinhaled particles and hence is an ideal sentinel for air quality.Macrophages upon stimulation have a characteristic “respiratory burst”.This is a manifest as a large increase in oxygen consumption and oxygenfree radical production. The free radicals inactivate toxins such asviruses and bacteria. Given their robustness and the rapidity of the“respiratory burst” responses to toxins, macrophages can serve as earlyresponders in the discrimination matrix 950.

An example of toxin discrimination by simultaneous monitoring ofmultiple metabolic signals according to the present invention followingthe exposure of some toxins is shown in FIGS. 17A and 17B. FIGS. 17A and17B illustrate how the discrimination of different analytes can berealized with a multi-sensor array of the present invention, and how onecan deduce which metabolic pathways are targeted by the agent. In FIG.17 a, it shows an example of the physiological signatures (energy andmetabolic) produced by the chemical Dinitrophenol (DNP), which uncouplesATPase synthesis from heat production and oxygen consumption. The resultis that to perform the same cellular processes, more oxygen, representedby line 1703, is consumed and more heat, represented by line 1701, andcarbon dioxide, represented by line 1702, are generated by lessefficient systems such as glycolysis. Glucose uptake, represented byline 1704, increases significantly, but lactate release, represented byline 1705, increases only slightly. Thus, the physiological signature ofDNP will be a rise of oxygen consumption and heat production. In thecase of cyanide in FIG. 17 b, the heat production 1711), glucose uptake1714, and lactate release 1715 increase while CO₂ production 1712 and O₂consumption 1715 decrease, respectively.

Generally in response to stress, the increase in heat production isdriven by an increase in the metabolic requirements of the cell. Thisincrease may be met by a general increase in oxygen consumption that isdriven by an increase in mitochondrial respiration and oxygenconsumption. The increase in caloric requirements can be met by afacilitation of glucose entry. The glucose can either enter theglycolytic pathway and be released as lactate or it can be completelyoxidized to carbon dioxide and water via mitochondrial respiration.Depending on the site of action of the toxin, and the cell type, one orboth pathways may be used. Some toxins (e.g., cyanide) targetmitochondrial respiration. Thus, despite adequate oxygen availability,the cell is unable to use oxygen to make ATP. Thus, glycolysis (glucoseconversion to lactate) serves a greater role in meeting the energydemands of the cell and the release of carbon dioxide is not longer theprimary fate of the glucose carbon and instead lactate release increaseswith glucose uptake. In contrast other agents such as DNP(dinitrophenol) decrease the efficiency of the mitochondrial processsuch that the oxygen requirements are greater for a given ATPaserequirement of the cell. The result is that the cell consumes moreoxygen and produces more heat to meet the ATPase demands. This maymanifest as a unique signature whereby oxygen consumption and heatproduction both increase. To meet the increase in energy demands,glucose uptake is increased. In this case as shown in FIG. 17 a, therewill be a corresponding increase in CO₂ production. However, that doesnot require the use of the inefficient glycolytic pathway to meet thecellular needs and lactate release will increase only slightly. Thus,unique biological signatures can be developed by tracing the time courseand amount of glucose uptake and subsequent oxidation and/or conversionto lactate in response to a given toxin.

FIGS. 18A and 18B displays the discrimination of toxins/agents bymonitoring characteristic temporal response of cellular phenotypestotoxins. Since the devices provided by the present invention are small,the temporal response is expected to be measured with millisecondresolution. Different agents such as toxins act on different time scalesthat will be used for discrimination. As an example shown in FIG. 18A,upon stimulation with endotoxin or phorbol esters, macrophages have anoxidative burst in which oxygen consumption, represented by line 1801,increases rapidly and markedly. Interestingly, this increase is not asdependent upon mitochondrial function as is seen in the liver. Inmacrophages endotoxin and phorbol esters activate a cytosolic enzyme(NADPH oxidase) that catalyzes the reaction (NADPH+2O₂→NADP⁺+2H⁺+2O₂ ⁻),Hydrogen peroxide (H₂O₂) is produced by dismutation of O₂, representedby line 1802. The free radicals generated in turn are cytotoxic due toits rapid conversion to OH⁻ and other radicals. Thus, the increasedconsumption of oxygen is less dependent upon a mitochondrial responseand is more rapid in onset and greater in magnitude that that of theliver, as shown below, the peak response is within five minutes and isparalleled by a rapid increase in free radical formation. In contrast,energy expenditure in hepatocytes, as shown in FIG. 18B, is increasedwhen challenged by agents, as shown by the increase in O₂ consumption1811. But this is accompanied by only a modest rise in free radicalproduction 1802, primarily mitochondrial in origin. Therefore, thecharacteristics of the biological signature can vary markedly both interms time of onset, rate of rise, magnitude, and deactivation rate ofthe individual metabolic or energy signature.

As displayed in these figures, the energy signature of the activatedmacrophage and the stimulated hepatocyte can be markedly different.Macrophages have a characteristically rapid increase in oxygenconsumption, which wanes despite the presence of the stimulus. Incontrast the hepatocyte exhibits a response that is slower in onset andsustained until the removal of the stimulus. The controlled addition ofa known amount of endotoxin will result in an increase in hepatic energyconsumption to support the very high metabolic activity of the liver.When unknown toxins are administered that uncouple or inhibit thisprocess, for example dinitrophenol or cyanide, the normal energydemanding functions of the liver, such as gluconeogenesis, arecompromised. The consequent cell specific change in metabolic activitycan be monitored and used as a canary to detect toxins.

FIGS. 19A and 19B show discrimination by characteristic responses in aconditioned environment. In particular, the difference in lactaterelease, represented by line 1901, following VX exposure is dependentupon preexposure to phenobarbital, with no preexposure shown in FIG. 19Aand preexposure in FIG. 19B, respectively. Preconditioning cells priorto toxin exposure can both serve to amplify a response to a toxin andhelp in the discrimination between toxins when the cells expose to morethan one toxin. Cultured hepatocytes have limited capacity to sustain agluconeogenic response to regulators of this process. However, priorexposure to dexamethasone markedly improves their response to the normalregulators. By enhancing the baseline gluconeogenic rate, specificinhibitory effects of toxins can readily be detected. Moreover, priorexposure to drugs, which enhance the metabolism of a cell, could be usedto discriminate agents. Organophosphates inhibit acetylcholinesteraseactivity. The data are shown in FIG. 10.

The nerve gas VX and other organophosphates inhibit glycolysis inneuroblastoma cells. However, pre-exposure of neuroblastoma cells toPhenobarbital may enhance the enzyme cytochrome P450, which in turn mayresult in the cellular conversion of these toxins to a more potent toxin(bioactivation). Thus following bioactivation of hepatocytes withPhenobarbital, the reduction of glycolysis with VX and parathion may bemarkedly enhanced.

FIG. 20 displays discrimination by characteristic reaction kinetics ofmetabolic pathways. The metabolic signatures of hepatocytes exposed to achange in glucose concentration in the absence, represented by line2001, and presence, represented by line 2002, of okadaic acid are shown,respectively.

Additionally, environment may be manipulated to determine where in agiven metabolic pathway the toxin is acting. This may be most effectiveif a toxin takes advantage of a metabolic signaling pathway to exert itsaction. One important pathway in cell signaling that is affected by anumber of toxins is the protein kinase A/cyclic AMP system. This systemwhen activated has profound and well-characterized metabolic responses,which include increases in gluconeogenesis and glycogen breakdown, andinhibition of glycolysis. As one example of pathway modulation, theresponse of a hepatocyte to an increase in the available glucose willdepend upon whether an agent blocks a particular enzyme pathway. Okadaicacid and microcystins inhibit protein phosphatase 1 and 2A activity.Okadaic acid has been shown to regulate cyclic AMP mediated events. Thusin the presence of okadaic acid, an increase in glucose concentrationwill not increase hepatic glucose uptake.

Other toxins may alter glycogen metabolism as well via their effect oncell signaling pathways. An increase in lactate release that isdisproportionate to the increase in glucose uptake, this would suggestan endogenous source of glucose (i.e., glycogen). Increases in lactaterelease disproportional to the increase in glucose consumption couldreflect toxins that either increases cyclic-AMP or intracellular calcium(e.g., organophosphates including the chemical warfare agents Sarin andVX). Increases in cyclic-AMP or calcium will activate hepatic glycogenphosphorylase, which will enhance glycogen breakdown. Since glycogen isa glucose polymer, upon its hydrolysis the cell will release glucose ormetabolize the glucose and release it as lactate. By applying specificmeasurements of intracellular calcium and calcium conductance, forexample, one may combine knowledge of metabolic pathways with specificmanipulations that allow one to dissect how a specific toxin exerts itsaction on metabolism.

FIG. 21 shows the shape of the action potential of excitable cells as anintegral sensing concept to access the physiological state of the cell.The control action potential of a cardiac cell, represented by line2101, and the action potential following agent washout, represented byline 2102, are a factor of four longer in duration than the actionpotential in the presence of Soman, represented by line 2103. Thus,measurement of the shape of the transmembrane action potential can serveas a sensitive but non-specific indicator of the effect of an agent on acell, and it may even allow discrimination between agents. The actionpotential can also be used as an initial way to detect the influence ofthe toxin agent on ion channels and ion pumps.

The effect of Soman on an action potential of a neuron is shown as anexample. The course of the action potential depends on the properfunction of various ion channels and functionally associated enzymes.Block of individual channels, like sodium channels with TTX, results inan immediate change of the action potential, which is easily detected.Likewise, blocking an associated enzyme, like the Na/K exchanger withOubain, results in a marked change of the action potential. Blocking theCa-channel in cardiac tissue with verapamil changes the shape of theaction potential dramatically, which could be extracted from the dataand being reflected in the action potential duration. For neural cellsmore so than for cardiac cells, the fitting of a Hodgkin-Huxley-typemodel to the observed action-potential shape can be used to estimate theconductance variation of key channels.

Thus, among other things, the present invention provides a matrix ofbiological signatures that can be used to define an orthogonal set ofcell lines, assays, and measurements for detecting previously known orunknown toxins, determining mechanisms of toxin activity throughreal-time biochemistry and autonomous hypothesis generation and testing,and using a bio-silicon circuit to specify and deliver the appropriateantitoxin for cellular-level defense. Additionally, the matrix ofbiological signatures can be integrated into a field deployable,configurable, and fully automatic device to detect a large number oftoxic agents with unsurpassed sensitivity, which does not require thedevelopment of specific assays to new toxin threats. Again, thisgenerality arises because the present invention allows measuring thebiological impact of toxins rather than the toxins themselves.

Example 2 Nanophyiometer and Bioreactor

In one aspect, the present invention relates to an apparatus formonitoring the status of a cell, more particularly, for screeningphysiological and biochemical effects of one or more cells on thenanoliter to picoliter scale. Such an apparatus according to the presentinvention may be termed as a Nanophysiometer, which in no way shouldlimit the scope of the invention.

FIG. 7 schematically shows a first embodiment of a Nanophysiometeraccording to the present invention. In FIG. 7, device 700 has a sensingvolume 704 filled with a solution of media containing a single ormultiple cells 701. The solution of media in the volume 704 can bemodified or changed using an inflow channel 708 and an outflow channel707, which are parts of a channel 721 that is in fluid communicationwith a supply or reservoir of media (not shown). The flow in each of thechannels 707, 708 can be controlled by valves 703, individually or incooperation.

The volume 704 is bounded on one side by a flexible membrane 705 thatcan be deflected, e.g., by pressurizing a closed volume 715 below theflexible membrane 705 through the channels 710 or 711, which are partsof a channel 723 that is in fluid communication with a supply orreservoir of fluid such as an air pump (not shown). The flow in each ofthe channels 710, 711 can also be controlled by valves (not shown),individually or in cooperation. Channel 723 is defined by a firstsubstrate 731.

The volume 704 is bounded on the other side by a second substrate 733having a first surface 735 and a second surface 737. The second surface737 of the second substrate 733 and the first substrate 731 defines thechannel 721. Several sensors 702 are positioned on the second surface737 of the second substrate 733 to measure the concentration of analytesin the sensing volume 704. The sensors 702 could be thin film electrodesand can be used to measure various analytes in the sensing volume 704 tomonitor the status of the cell 701. The sensors 702 are coupled throughleads 709 to a sensing unit (not shown), respectively. Note that bydeflecting the membrane 705 forces may be applied to the cell 701, inparticular when the cell 701 is attached to the membrane 705, so thatthe response of the cell 701 to the applied force can be detected.Further note that device 700 can be utilized to grow a cell. Forinstance, a cell can be attached to the membrane 705, and the status ofthe cell can be monitored by sensors 702.

FIG. 12 schematically shows a second embodiment of a Nanophysiometeraccording to the present invention. In FIG. 7, device 1200 has a systemsupport structure 1201 beneath a microfluidic channel 1202. Themicrofluidic channel 1202 is formed in a micromachined substrate 1205. Anumber of wells 1251 are in fluid communication with the microfluidicchannel 1202. Epoxy 1203 provides a fluid-tight seal between the supportstructure 1201, the micromachined substrate 1205, and a cover 1204.Cover 204 is transparent and supports an array of sensors 1206.Individual electrochemical sensors 1206 include enzyme-activatedelectrodes, and enzyme electrodes that can determine the extracellularfluidic composition and the consumption and release of metabolicsubstrate and byproducts when used in combination withsilver/silver-chloride reference electrode 1207, gold counter electrode1208 and an amperometric or potentiometric instrument 1215 that measuresand/or applies voltages and/or currents for the combination ofelectrodes 1206, 1207, 1208. Oxygenated perfusate reservoir 1209, oxygensupply for the perfusate oxygenator, computer-controlled nanolitersyringe pump 1213 and check valves 1211 and 1212 allow the withdrawal bythe pump 1210 of oxygenated perfusate 1213, and its subsequent injectionthrough tubing 1214, to the microfluidic channels 1202.

Cells are placed into the wells 1251. Each well has a volume of lessthan 1 nL and may receive one or more cells to be confined therein. Theelectrochemical sensors 1206, 1207, 1208 monitor the metabolic state ofeach cell or cells. The microchannels 1202 with cross-sections on theorder of 10 μm×10 μm supply analytes to the cells, remove waste, andallow for the introduction of biological agents into the wells. One toseveral cells can be placed into each well with a micropipette orthrough the fluid channels. Among other things, device 1200 has externalpumps and valves for automated control of the flow and introduction ofthe analytes. Moreover, device 1200 has planar electrochemical sensors1206, and nanoLiter sized volumes 1251 resulting in high sensitivity andfast response times. Additionally, device 1200 has on-chip sealed wellsand channels for cell storage, delivery of analytes and biologicalagents, and removal of waste. Utilization of sensors 1206, 1207, 1208having different electrochemical characteristics allows formultispectral readout. The transparency of the cover 1204 also makesoptical detecting available.

Referring now to FIG. 13, a third embodiment of a Nanophysiometeraccording to the present invention is shown. In FIG. 13, device 1300 hasboth external valve actuators and on-chip pumps. Micromachined substrate1320, which can be formed in silicon, glass, ceramic, plastic, orpolymer, defines microfluidic channels 1301, which can be used as oxygeninlet for oxygenating the perfusate interfacing with a cell 1312. Anoptional cover slip 1302 covers the microfluidic channels 1301.Addressable piezoelectric nanoactuator array 1307 is supported by anactuator platform (or substrate) 1305 and support posts 1304. Checkvalves 1311 allow the withdrawal of oxygenated perfusate from thereservoir 1303 and its injection into the microfluidic channels 1301 andsample wells 1308 that contain the living cells 1312. The chambercorresponding to each well 1308 is drained by microfluidic line 1310.Sensors in the form of interdigitated microelectrodes 1309 allow theelectrochemical determination of analytes in each chamber 1308. Opticaldetectors (not shown) can also be utilized through the cover 1302, whichis at least partially transparent.

Device 1300 shows how external pumps utilized in device 1200 as shown inFIG. 12 can be replaced with on-chip pumps making the device astandalone unit. Device 1300 could be match box sized incorporating,wells, sensors, pumps and actuators to achieve the goal of massivelyparallel testing. On-chip pumps can be a microscale version of thesyringe pumps used in device 1200. Using standard microfluidictechnology, each pump will, for example, may have a reservoir coveredwith a flexible membrane. This membrane will be moved in or out bychanging the length of a piezoelectric element. An array of individuallyaddressable piezoelectric filaments can be utilized to provide separateactuation of multiple pumps. This actuator ‘bed-of-nails’ may alsoprovide valving by pinching closed sections of the channels between thereservoirs.

An exemplary electrode can be utilized to practice the present inventionincluding utilization in any embodiment of Nanophysiometer is shown inFIG. 26. FIG. 26A is a photomicrograph of the electrode array withplatinum, iridium oxide, and platinum microstrips on a glass substrate.FIG. 26B shows a pH calibration of the sensor. Such an Iridium oxide pHelectrode can be used to form on a platinum interdigitatedmicroelectrode array.

Referring now to FIG. 27, a fourth embodiment of a Nanophysiometeraccording to the present invention is shown. In FIG. 27, device 2700 hasa first substrate 2721 and a second substrate 2723 defining a sensingvolume 2704 therebetween. The sensing volume 2704 contains a single ormultiple cells 2701 in sufficient close proximity to sensors 2702designed to monitor the physiological status of the cell or cells 2701such that any measurement related to the physiological status of thecell or cells 2701 can be made at a time period shorter than acharacterization time corresponding to the physiological status of thecell or cells 2701. The sensing volume 2704 is in fluid communicationwith a channel 2725, which has an inlet portion 2707 and an outletportion 2708. The liquid media in the sensing volume 2704 can berefreshed or adjusted using the inlet 2707 and outlet 2708, which inturn are controlled by valves 2703, respectively. Measured signals fromthe sensor(s) 2702 can be read out through a connection 2709 toelectronics such as a controller (not shown). Additional channels intothe sensing volume 2710 can be used to deliver agents and other analytesto the sensing volume 2704. The second substrate 2723 may be at leastpartially transparent for optical detecting. The device 2700 can beformed, for example, by fusing a first part containing the sensors and asecond part containing the fluidic channel and the valves together.

Referring now to FIG. 28, a fifth embodiment of a Nanophysiometeraccording to the present invention is shown. In FIG. 28, device 2800 hasan input channel 2808, an outlet channel 2807 and an additional channel2809, which may be used as an additional outlet channel or to flush outthe contents from a sensing volume 2802 that is in fluid communicationwith each of channels 2807, 2808, and 2809. The liquid media in thesensing volume 2802 is changed or adjusted either continuously or in astop flow fashion through actuation of the valves 2805 in the inlet 2808and outlet channel 2807 by pressurizing the media in the inlet channel2808, respectively. The valves 2805 are actuated through lines 2803,each being in fluid communication with a supply or reservoir of fluidsuch as pressured air. The inlet channel 2807 is equipped with a seriesof valves 2805 which can be actuated to act as a peristaltic pump. Thesame can be used for the outlet channel 2807. The sensing volume 2802 isequipped with multiple sensors 2801 to monitor the physiological statusof a single cell or cells 2804. The sensors 2801 can take various forms.For examples, sensors 2801 can be in the form of functionalized thinfilm metal electrodes that are positioned in sufficient close proximityto the cell or cells 2804. The surface of each sensor may have a coatingthat facilitates cell adhesion (not shown). The device 2800 can beformed, for example, by fusing a first part 2815 containing the sensorsand a second part 2816 containing the fluidic channel and the valvestogether.

Referring now to FIG. 29, a sixth embodiment of a Nanophysiometeraccording to the present invention is shown. In FIG. 29, device 2900 hasa valveless structure to trap or confine a cell 2907 in the sensingvolume 2906 containing liquid media. The cell 2907 is placed in thesensing volume 2906 by flowing the cell 2907 in through an input channel2903. The sensing volume 2906 is in fluid communication with an outputchannel 2905, which is optional and has a cross section smaller thanthat of the input channel 2903. Another channel 2904, which is in fluidcommunication with the sensing volume 2906, may be used to deliver thecell 2907 to the sensing volume 2904, or to remove the cell 2907 at theend of the measurement. The sensing volume 2906 is adapted such that itis not very much bigger than the size of the cell 2907 so only one or asmall number of cells may enter the sensing volume 2906 at one time. Inoperation, once a cell or cells are placed in the sensing volume 2906through a loading phase, the media in the channel 2903 is changed to amedia corresponding to a measurement phase or maintenance phase. Afterthe measurement phase, the channel 2903 can be pressurized to reversethe flow of the media to eject the cell 2907 from the sensing volume2906. The media of the sensing volume 2906 can be exchanged or adjustedeither by diffusion from the input/output channel where a constant flowis of fresh media for maintenance or by appropriately dimensioning theadditional output channel 2905 which leads to a flow away from thesensing volume 2906. Multiple sensors 2901 are positioned in the sensingvolume 2906 to monitor the physiological status of a single cell orcells 2907. The sensors 2901 can take various forms. For examples,sensors 2901 can be in the form of functionalized thin film metalelectrodes that are positioned in sufficient close proximity to the cellor cells 2907. The surface of each sensor may have a coating thatfacilitates cell adhesion (not shown). The device 2900 can be formed,for example, by fusing a first part 2912 containing the sensors and asecond part 2910 containing the fluidic channel and the valves together.

Referring now to FIG. 30, a seventh embodiment of a Nanophysiometeraccording to the present invention is shown. In FIG. 30, device 3000 canbe considered as a multi-trap version of the valveless nanophysiometeras shown in FIG. 29 and has a plurality of sensing volumes 3006, 3016,3026 in an array with a common inlet channel 3003 and outlet channel3006. More sensing volumes can be introduced. The additional channels3007 lead to a common channel 3017, which also has an inlet channel 3004and an outlet channel 3005. The sensors 3001 can be read outindividually, or in cooperation, from each sensing volume containing oneor more cells 3002, respectively.

All the embodiments of a Nanophysiometer according to the presentinvention shown above can be utilized, among other things, to monitorthe status of a cell that consumes or produces energy. The energyconsumption or production of the cell includes consumption of a chemicalcomponent by the cell that relates to the metabolic status of the cell,where the chemical component can be any of pH, K, oxygen, lactate,glucose, ascorbate, serotonin, dopamine, ammonina, glutamate, purine,calcium, sodium, and potassium. As an example, FIG. 31 shows theutilization of a Nanophysiometer according to the present invention, inparticular, according to the embodiment shown in FIG. 28, to measuretemporal response of cell or cells to changes in pH and oxygen.

More specifically, in FIG. 31A, without bounding to any theory, curve3100 represents average pH as a function of time in a 100 pL wellcontaining a single cell with no flow, based upon the assumptions thatthe well solution is initially at a pH of 7.2, and at time t=0, the cellbegins producing lactate at a constant rate of approximately 6×10⁻¹⁰mmol/cell/hr. Thus, for a single cell in the well, there areapproximately 2×10⁻¹³ mmol of lactate produced per second. Finally, itis assumed that there is one proton produced for every lactate moleculeproduced. For a pH close to neutral this is a reasonable estimate, butin reality the ratio of protons to lactate molecules goes down as pHdeparts from neutral, so this model may overestimate the pH change,which, however, would limit the scope or validity of the presentinvention.

In FIG. 31B, curve 3102 represents the same data as shown in FIG. 31A,except it is plotted as a function of logarithmic time to show that theresponse is constant until the protons have time to diffuse from thecell to the electrode, which can be characterized by a diffusion time ordiffusion constant. A good measurement should be done within a timeperiod that is shorter than the diffusion time.

Note that the pH response of a system can be characterized by the timeit takes for the pH to drop by a certain amount. For FIG. 31C, theinitial pH is 7.2 and the “target” pH is 7.0. Using the model presentedin FIGS. 31A and 31B, the time it takes for the pH to drop by 0.2 is alinear function of the system volume, as illustrated in the plot—a pLvolume requires about 0.2 msec, whereas a microliter volume takes about3 minutes.

Table 2.1 gives a list of events and corresponding characterizationtimes for the events to take place. As one can see, some events relatedto changes in the metabolism of cells happen in a few milliseconds.

TABLE 2.1 Characterization Events Time (Seconds) 1. Mixing time tohomogenize liquid in a large-scale 10⁴-10⁸ bioreactor (10-100 m³) 2. 90%liquid volume exchange in a continuous 10⁵-10⁶ reactor 3. Oxygentransfer (forced not free diffusion) 10²-10³ 4. Heat transfer (forcedconvection) 10³-10⁴ 5. Cell Proliferation, DNA replication 10²-10⁴ 6.Response to environmental changes (temperature, 10³-10⁴ oxygen) 7.Messenger RNA synthesis 10³-10⁴ 8. Translocation of substances intocells 10¹-10³ (active transport) 9. Protein synthesis 10¹-10² 10.Allosteric control of enzyme action 10⁰    11. Glycolysis 10⁻¹-10⁻² 12.Oxidative phosphorylation in mitochondria 10⁻² 13. Intracellularquiescent mass & heat transfer 10⁻⁵-10⁻³ (dimension 10-5 μm) 14.Enzymatic reaction and turnover 10⁻⁶-10⁻³ 15. Bonding between enzyme &substrate, inhibitor 10⁻⁶ 16. Receptor-ligand interaction 10⁻⁶

For further comparison, Dn=Diffusion time calculated for Oxygen, wheren=25, and lactate n=5, for spherical geometry (indexed as “s”) and cubicgeometry (indexed as “c”), respectively, is given below:

D 5 := 5 ⋅ 10⁻¹⁰ ⋅ m² ⋅ s⁻¹, D 25 := 25 ⋅ 10⁻¹⁰ ⋅ m² ⋅ s⁻¹i := 0  …  10,  x_(i) := 10^(−i) ⋅ m${t\; 5_{i}}:={{\frac{\left( x_{i} \right)^{2}}{{2 \cdot D}\; 5}\mspace{34mu} t\; 25_{i}}:=\frac{\left( x_{i} \right)^{2}}{{2 \cdot D}\; 25}}$${V_{s_{i}}:=\frac{4 \cdot \left( \frac{x_{i}}{2} \right)^{2}}{3}};\mspace{11mu}{V_{c_{i}}:=\left( x_{i} \right)^{3}}$

$x = {{\begin{matrix}1 \\0.1 \\0.01 \\{1 \cdot 10^{- 3}} \\{1 \cdot 10^{- 4}} \\{1 \cdot 10^{- 5}} \\{1 \cdot 10^{- 6}} \\{1 \cdot 10^{- 7}} \\{1 \cdot 10^{- 8}} \\{1 \cdot 10^{- 9}} \\{1 \cdot 10^{- 10}}\end{matrix}m\mspace{45mu}{t5}} = {{\begin{matrix}{1 \cdot 10^{9}} \\{1 \cdot 10^{7}} \\{1 \cdot 10^{5}} \\{1 \cdot 10^{3}} \\10 \\0.1 \\{1 \cdot 10^{- 3}} \\{1 \cdot 10^{- 5}} \\{1 \cdot 10^{- 7}} \\{1 \cdot 10^{- 9}} \\{1 \cdot 10^{- 11}}\end{matrix}s\mspace{50mu}{t25}} = {\begin{matrix}{2 \cdot 10^{8}} \\{2 \cdot 10^{6}} \\{2 \cdot 10^{4}} \\200 \\2 \\0.02 \\{2 \cdot 10^{- 4}} \\{2 \cdot 10^{- 6}} \\{2 \cdot 10^{- 8}} \\{2 \cdot 10^{- 10}} \\{2 \cdot 10^{- 12}}\end{matrix}s}}}$

${{Vs} = {{\begin{matrix}166.667 \\0.167 \\{1.667 \cdot 10^{- 4}} \\{1.667 \cdot 10^{- 7}} \\{1.667 \cdot 10^{- 10}} \\{1.667 \cdot 10^{- 13}} \\0 \\0 \\0 \\0 \\0\end{matrix}{liter}\mspace{45mu}{Vc}} = {\begin{matrix}{1 \cdot 10^{3}} \\1 \\{1 \cdot 10^{- 3}} \\{1 \cdot 10^{- 6}} \\{1 \cdot 10^{- 9}} \\{1 \cdot 10^{- 12}} \\{1 \cdot 10^{- 15}} \\0 \\0 \\0 \\0\end{matrix}{liter}}}}\mspace{25mu}$

-   Oxygen noise 10 micromolar, sensitivity scales with area-   Glucose noise 100 micromolar

${tr}:=\frac{\left( \left( {6.5 \cdot {mm}} \right) \right)^{2}}{{2 \cdot D}\; 25}$

-   Flux at surface; signal-to-noise scales with area:

tr = 140.833  min  $F:=\frac{1}{n \cdot e \cdot F \cdot A}$(.000100  m)³ = 1 × 10⁻⁹  liter

-   Oxygen in water at 40° C.

D := 0.0000324  cm² ⋅ s⁻¹$D = {3.24 \times 10^{- 9}\mspace{20mu}\frac{m^{2}}{s}}$

FIG. 31D displays the results of the test of the Nanophysiometer with aplatinum interdigitated array that senses oxygen. The microfluidicnanophysiometer 3101 is similar to the one as shown in FIG. 28. Theoxygen sensing electrodes 3102 are coupled to a potentiostat 3103 andthe computer 3104 that generates plots of the oxygen concentration as afunction of time for fluid that is oxygen saturated 3105, perfused withambient-oxygen 3106, or nitrogen sparged 3107. The rapid response 3108shows that these electrodes can track oxygen changes that occur in tensof milliseconds, which is possible because, among other advantages, thedevice(s) of the present invention has sensor(s) positioned sufficientlyclose to the cell, i.e., at nano-scale dimension. In other words, thesmall (in term of dimension) is fast (in term of response), and the fastis better (in term of quality of signals, and thus applications).

FIG. 31E displays as an example an individually addressableinterdigitated microelectrode array that can be used to practice thepresent invention. A 2 mm wide pad 3101 of platinum on the glasssubstrate is coupled to interdigitated microstrip electrodes 3102 and3103 that are five microns wide and separated by five microns and thusforming a plurality of fingers. Each of the individual fingers can becoated with silver/silver chloride, gold, iridium oxide, or enzymes todetermine what each microstrip may detect.

Example 3 Improved Sensor Head

In one aspect, the present invention relates to a device as shown inFIGS. 11(A)-(C) for detecting at least one analyte of interest eitherproduced or consumed by at least one cell or cells 1107, wherein the atleast one cell or cells 1107 is placed in a chamber 1128. In oneembodiment of the present invention as shown in FIGS. 11(A)-(C), adevice or a sensor head 1100 includes a body portion 1151 and asubstrate 1153 defining a chamber 1128. The body portion 1151 can becircular, oval, square, or any other geometric shape cross-sectionally.In the embodiment shown, the body portion 1151 has a circular crosssection. The substrate 1153 has a first surface 1155 and an opposite,second surface 1157. A membrane 1127 is positioned on the first surface1155 of the substrate 1153. The membrane 1127 is partially transparentto allow optical signals passing through. For instance, in oneembodiment as shown in FIGS. 11(A)-(C), the membrane 1127 comprises aSi/SiN membrane. An insert 1129 that contains the living cells 1107 isplaced into the chamber 1128 and is sealed to the body portion 1151 byan O-ring 1136 that fits into a corresponding O-ring groove 1137 formedon the body portion 1151.

An inlet 1101 is in fluid communication with the chamber 1128 through anend portion 1122. Inlet 1101 may also be in fluid communication with oneor more reservoirs of mediums (not shown), where each medium may containa different analyte of interest. The device 1100 also has a firstelectrode 1124 having a first electrochemical characteristic, and asecond electrode 1126 positioned away from the first electrode 1124 andhaving a second electrochemical characteristic. The device 1100 mayfurther have a reference electrode 1125. In cooperation with thereference electrode 1125, the first electrode 1124 can detect a firstanalyte of interest either produced or consumed by at least one cell orcells 1107, and the second electrode 1126 can detects second analyte ofinterest by at least one cell or cells 1107, respectively andsimultaneously. Alternatively, in cooperation with the referenceelectrode 1125, the first electrode 1124 and the second electrode 1126can detect one analyte of interest either produced or consumed by atleast one cell or cells 1107 in the chamber 1128. Analytes of interestcan be introduced to the chamber 1128 through the inlet 1101, 1122. Anoutlet 1104 is in fluid communication with the chamber 1128 through anend portion 123 for introducing medium away from the chamber 1128.

The device 1100 may utilize an amperemeter electrically coupled to thefirst electrode 1124 and the second electrode 1126 for detecting acurrent as a function of the two analytes of interest either produced orconsumed by at least one cell or cells 1107 in the chamber 1128.Alternatively, the device 1100 has a potentiostat 1103 electricallycoupled to the first electrode 1124 and the second electrode 1126 fordetecting a voltage as a function of the two analytes of interest eitherproduced or consumed by at least one cell or cells 1107 in the chamber1128. Meters such as potentiostat 1103 can be further interfaced to adata acquisition computer so as to save, process and analyze detectedsignals.

Moreover, the device 1100 may further have additional electrodes, eachhaving a different electrochemical characteristic to one of the firstelectrode 1124 and the second electrode 1126 and being positioned awayfrom the first and second electrode 1126 s. For examples, the device1100 may have a third electrode 1146 positioned away from the firstelectrode 1124 and the second electrode 1126. In the embodiment as shownin FIGS. 11(A)-(C), the first electrode 1124 is a gold electrode and thesecond electrode 1126 and the third electrode 1146 both are a platinumelectrode. Moreover, Additionally, the first electrode 1124 has a crosssection larger than that of both the second electrode 1126 and the thirdelectrode 1146, which are substantially similar to each other for theembodiment as shown (they are indeed platinum wires). Using the platinumelectrodes 1126 and 1146 as a counter electrode, the device 1100 addsthe ability to perform electrochemical and spectrochemical analysiswithin the sensor head 1100. Of course, the first electrode 1124, thesecond electrode 1126 and the third electrode 1146 each can havedifferent surface film, coating, shape, material modifications toaccommodate the needs for detecting one or more desired analytes ofinterest.

Furthermore, the device 1100 has a fiber-coupled optical system 1102that has a first end 1162, a second end 1164 and an optical fiber bodyportion 1166 defined therebetween. The first end 1162 of the opticalfiber body portion 1166 reaches in the chamber 1128 capable of detectingan optical signal related to the analytes of interest either produced orconsumed by cell or cells 1107. Thus, the fiber-coupled optical system1102 can monitor fluorescence of the cells by light 1121 emitted intothe chamber 1128.

Thus, the device 1100 with the electrodes embedded in a chemicallystable epoxy, can measure oxygen, glucose, lactate andoxidation-reduction potential in addition to the pH measurement that iscurrently available from the membrane 1127 in the bottom of the chamber1128 and illuminated from below through an optical window 1138, to forma light-addressable potentiometric sensor. The fiber-coupled opticalsystem 1102 can use autofluoresence to measure intracellular NADH/NADratios and voltage and calcium-sensitive dyes to determine transmembranepotential and intracellular calcium. The ability of all sensors tofunction simultaneously allows the specification of a self-consistentset of metabolic fluxes.

Moreover, in one embodiment as shown in FIG. 14, the fiber-coupledoptical system 1102 can be coupled to an optical detector 1400. Theoptical detector 1400 has an optional cover slip member 1420 having afirst surface 1421 and a second surface 1423, wherein the first surface1421 of the cover slip 1420 is underneath the chamber 1428 in contactwith substrate 1408, and the second surface 1423 of the cover slip 1420is optically coupled to a first end 1422 of an optical fiber 1403. Inone embodiment as shown in FIG. 14, the cover slip member 1420 mergeswith the first end 1422 of the optical fiber 1403. A light source 1402optically coupled to a second end 1424 of the optical fiber 1403. A beamsplitter 1404 is optically coupled to the optical fiber 1403 andpositioned between the light source 1402 and the cover slip 1420 fordirecting optical signals transmitted through the optical fiber 1403corresponding to the optical response from a first direction to a seconddirection. And the optical detector 1400 further has an analyzer 1401for receiving the optical signals directed by the beam splitter 1404.

In operation, monochometer 1401 selects wavelength of light to bemeasured by the photodetector 1409. The light source 1402 is coupled toan optical fiber 1403 and the dichroic beamsplitter 1404 that deliverslight to the chamber 1428 where a droplet of perfusate 1405 containingat least one cell 1406. The fiber 1403 is coupled through thetransparent substrate 1408, which is supported by sidewalls 1407, toobtain the signals regarding the status of the cell 1406.

Note that while the optical detector 1400 is discussed here inconnection with a sensor head, the optical detector 1400 can be readilyutilized with devices disclosed in other examples of the presentinvention including the NanoPhysiometer, the well plates, theMicrobottles, and the Picocalorimeter.

Additionally, the optical detection method and instrument of the presentinvention can be combined with any of the sensors disclosed in thisspecification. The optical detection method and instrument uses anoptical fiber technique to illuminate the wells and to extract thefluorescence and luminescence signals. Imaging an entire cell onto asingle sensor element offers greatly enhanced signal-to-noise ratios,among other things.

Perhaps with the exception of NADH/NADPH autofluorescence, the opticaldetection method and instrument may need the introduction of somefluorescent probes into the cell. Some fluorescence dyes do not requiredirect intracellular access and can be directly incorporated in thesensing platform and read out with the fiber optics system disclosedherein. Optical dyes could be administered and purged through thefluidics channels already incorporated in the cell physiometer.

To enhance the efficiency of wavelength separation, one may useBragg-filters embedded in the optical fibers. Light indicator can eitherutilize a photomultiplier or a photodiode.

Example 4 Microbottles

In one aspect, the present invention relates to a device 500 formonitoring status of cell 501 or cells as shown in FIG. 5. In oneembodiment as shown in FIG. 5, a device 500 includes a first substrate550 having a first surface 551 and an opposite second surface 553. Thedevice 500 further has a second substrate 560 supported by the firstsubstrate 550. The second substrate 560 has a first surface 561, anopposite second surface 563, a body portion 502 between the firstsurface 561 and the second surface 563, a first side surface 565 and anopposite second side surface 567, wherein the body portion 502 defines afirst passage 511 between the first side surface 565 and the second sidesurface 567 and an opening 569 on the first surface 561 of the secondsubstrate 560 and in fluid communication with the first passage 511.Sidewalls 571, 573, 575 are positioned above the first surface 561 ofthe second substrate 560. The second substrate 560 can be made fromsemiconductor or insulating materials. In one embodiment, the secondsubstrate 560 is made from silicon.

The device 500 also includes a third substrate 580 having a firstsurface 581 and an opposite second surface 583. Sensors (not shown) canbe added to the second surface 583 of the third substrate 580 having afirst surface 581 and an opposite second surface 583 to measure theconcentrations of analytes in the extracellular fluid 515 of chamber590, as can optical sensors 1400 in FIG. 14, to measure intracellularand transmembrane physiological signatures as discussed above of thecells 501 in chambers 590. The third substrate 580, the sidewalls 571,573 and the second substrate 560 define a chamber 590 that is in fluidcommunication with a second passage 591 defined by portions of thesidewall 571 and the third substrate 580. The second passage 591 is influid communication with a supply or reservoir of a medium (not shown).As it is shown, optionally, the third substrate 580, the sidewalls 573,575 and the second substrate 560 define another chamber 592 that is influid communication with a third passage 595 defined by portions of thesidewall 575 and the third substrate 580. The third passage 595 is influid communication with a supply or reservoir of a medium (not shown).The chambers 590 and 592 are in fluid communication through a passage593 located therebetween. The device 500 further includes a pair offirst controls 509 a, 509 b positioned inside the first passage 511 forcontrolling the flow of a medium through the first passage 511corresponding to chamber 590. Additional first control 509 c can beutilized to control the flow of a medium through the first passage 511corresponding to chamber 592 with or without first control 590 b. Firstcontrols 509 a, 509 b, and 509 c can work in any pair, in group, orindividually. Note that although the device 500 is shown to have a twochamber structure in this embodiment, it can alternatively have a singlechamber structure or an N chamber structure, where N is an integergreater than two.

The device 500 further includes at least one sensor 505 positioned inthe first passage 511 proximate to the opening 569, wherein a cell 501is positioned in the chamber 590. In one embodiment, the cell 501 issealed to the second substrate 560 by at least one gigaohm seal 503. Thecell 501 has a membrane 541 forming a substantially enclosed structureand defining an intracellular space 543 therein. The intracellular space543 of the cell 501 is in fluid communication with the first passage 511through the opening 569 of the second substrate 560.

The membrane 541 of the cell 501 defines an opening 549 through whichthe intracellular space 543 of the cell 501 is in fluid communicationwith the first passage 511 through the opening 569 of the secondsubstrate 560. The device 500 further includes a punching element 506positioned underneath the opening 569 of the second substrate 560 formaking the opening 549 on the membrane 541 of the cell 501. The punchingelement 560 can be a mechanical device such as a pressure-based suctiondevice (not shown) or an electroporation device such as an electricpotential sucking device.

As such formed, the device 500 allows cells with intracellular andextracellular spaces in fluid communication through microfluidicchannels such as passages 511, 591, 593, 595.

In one operation mode, when a first medium is introduced into the firstpassage 511, the intracellular space 543 of the cell 501 is in fluidcommunication with the first passage 511 with the first medium, thesensor 505 measures the response of the cell 501 to the first medium.The response can be viewed as an intracellular response to the firstmedium, which may contain agent or agents. The measured signals can beamplified by amplifier 512 to generate an output 513 and/or transmittedto a controller 508 as a feedback, which in turn can control the flow ofthe first medium through fluid control 509 b (and 509 a, 509 c). Thefirst medium can also be used to provide nutrition to the cell 501 andto maintain the cell 501 at a desired status.

In another operation mode, when a second medium is introduced into thechamber 590 through the second passage 591, at least part of themembrane 541 of the cell 501 is in contact with the second medium in thechamber 590, the sensor 505 measures the response of the cell 501 to thesecond medium. The response can be viewed as an extracellular responseto the second medium, which may contain agent or agents. The measuredsignals can be amplified by amplifier 512 to generate an output 513and/or transmitted to a controller 508 as a feedback, which in turn cancontrol the flow of the first medium through fluid control 521 a (and521 b). The second medium can also be used to provide nutrition to thecell 501 and to maintain the cell 501 at a desired status.

In yet another operation mode, when a first medium is introduced intothe first passage 511 and a second medium is introduced into the chamber590 through the second passage 591, respectively, the intracellularspace 543 of the cell 501 is in fluid communication with the firstpassage 511 with the first medium and at least part of the membrane ofthe cell 501 is in contact with the second medium in the chamber 590,the sensor 505 measures the responses of the cell 501 to the firstmedium and the second medium. From these measurements, the status of thecell 501 can be monitored.

If a plurality of sensors is utilized to practice the present invention,they can be substantially the same. Or, alternatively, at least two ofthem can be different from each other.

In another application, the device 500 can be utilized to control thephysiological status of at least one cell. Normally, a cell controls itsphysiological status through an internal cellular control mechanism. Inone embodiment, the device 500 can be used to provide at least onemedium to the cell 501 such that at least part of the membrane of thecell 501 is in contact with the medium to override the internal cellularcontrol mechanism.

In one operation mode, a first medium is supplied into the intracellularspace 543 of the cell 501 through the opening 569 in the membrane 541,and a second medium is supplied into the chamber 590 such that at leastpart of the membrane 541 of the cell 501 is in contact with the secondmedium. The response of the cell 501 to the second medium is measured,and the composition of the second medium is adjusted based on theresponse to affect the overriding of the internal cellular controlmechanism. Moreover or alternatively, the response of the cell 501 tothe first medium is measured, and the composition of the first medium isadjusted based on the response to affect the overriding of the internalcellular control mechanism.

In another operation mode, the concentration of at least one selectedcomponent of the medium can be monitored and the composition of themedium can be adjusted based on the monitored concentration of at leastone selected component of the medium to affect the overriding of theinternal cellular control mechanism.

Still referring to FIG. 5, the device 500 alternatively can be viewed asto have a biolayer 510, a physical layer 520 and an infolayer 530. Thebiolayer 510 includes chamber 590 that can contain extracellular fluid515, a living cells 501 with its corresponding transmembrane ionchannels and ion-channel complexes 514 and pumps and transporters 515.The intracellular space 543 of the cell 501 through the opening 569 inthe membrane 541 is in fluid communication with a fluidic medium 507that functions as an artificial intracellular medium. The physical layer520 includes sensing electrodes 505, valves 509 a, b, c and otherelements such as punching element 506. The Infolayer 530 containsamplifiers 512, reconfigurable digital and analog software programmabledigital signal processors 508 and outputs 513. The membrane 514 issealed to the substrate 502 by the gigaohm seal 503. Microfluidicpassages/channels 511 and 509 a, b, c allow control of the fluidiccontents 507 of the medium and allow intracellular communication betweenmultiple, coupled cells. Valves 521 a, b, c allow extracellularcommunication between cells if needed.

The device 500 according to the present invention may be termed as a“microbottle,” which in no way should limit the scope of the presentinvention. FIGS. 3(A)-(C) shows another embodiment of the microbottleaccording to the present invention. In FIG. 3, device or microbottle 300has a biolayer 310, a physical layer 320 and an infolayer 330. Thebiolayer 310 includes a cellular biological membrane or synthetic lipidmembrane 301 containing ion channels or ion-channel/receptor complexes314 and pumps and transporters 315, such that the inner surface 304 ofthe membrane 301 is exposed to an fluidic medium 307 that functions asan artificial intracellular medium. The physical layer 320 includes themicrobottles, picocalorimeters, microfluidics 305, 307, andsensor/electrodes 310. The infolayer 320 contains amplifiers 312,reconfigurable digital an analog software programmable digital signalprocessors (“DSPs”) 308 and the system output 313. The microbottle 300has a silicon substrate 302, and the membrane 301 is sealed to thesubstrate 302 by the gigaohm seal 303. Microfluidic channels 311 andvalves 309 allow control of the content of the fluidic contents 307 ofthe container and allows intracellular communication between multiple,coupled membranes 301.

FIG. 4 shows yet another embodiment of the microbottle according to thepresent invention. In FIG. 4, a living cell 401 is maintained in achamber 412 whose fluidic contents are maintained by valves 404, whichare connected to perfusate reservoirs 411 by microfluidic channels 410.A controller 406 is coupled by sensing leads 407 to sensors 405, 409 and413 to sense the chemical composition in the extracellular space 402 andthe chemical composition and/or state of internal organelles and/ornatural or artificial markers 414 in the intracellular space 403.Through control leads 408, the controller 406 then adjusts the valves404 to maintain the proper extracellular environment and the level oftoxin or agents in the reservoirs 411 to which the cell 401 is exposed.The controller 406 can also control the exposure of the cell 401 tolight by means of a controlled light source 418 that can be used toalter the conductance of transmembrane ion channels 416 or pumps ortransporters 417.

Accordingly, the microbottle provided by the present invention invarious embodiments can provide direct interface for measuring andcontrolling ion concentrations on both sides of a cell or syntheticmembrane. The microbottle according to the present invention can beadapted to a variety of applications where the biological element is anactive component in the circuit design. The microbottle can be used as asensing element and allows the release of its content or in case of acellular cap simulates the cell change cell function. In one embodimentof the present invention, as shown in FIG. 3, the microbottle has asilicon base layer as the focus of the sensing, signal processing andlogic, a structural assembly for supporting microfluidics for deliveryof ionic species, and a membrane, which is a biologically activeelement.

As illustrated in FIG. 3, in one embodiment according to the presentinvention, the microbottle 300 has a layered structure, where each layercan be fabricated individually and assembled in a flip chip manner byconventional bonding techniques. The base layer 310 is a silicon waferthat can be fabricated in conventional CMOS technique containing thesensing elements, actuators, the readout electronic and the logicelements to combine the microbottle in an array structure. Themicrofluidics layer 330 includes containers/chambers interconnected withchannels. The fluids in the channels can be controlled throughmicrovalves and pumps. Each chamber is sealed with an active membranecap. The membrane could be either biofunctional or biomimetic.

Silicon Base Layer: Microelectronic sensors can be chosen for themeasurement of Microbottle input and output parameters. They can be usedto control the physio-chemical parameters in the silicon container andthe environment of hermetically sealed devices. They can also be used todetect changes of the cellular behavior in response to an experimentaltreatment.

The basic sensor types to monitor the container content of microbottle,among other things, may include microelectrodes, electrochemicalsensors, amperimetric sensors, potentiometric sensors, oxygen and otherelectrochemical sensors and field effects transistors (FET), where thegate electrode is made of or is coated with an electrochemically activematerial. This material can affect the source/drain current by bindingcharge from the contents in the microbottle to its surface, creating avoltage drop across the gate insulator. FET-based sensors can be usedfor different measurement tasks. The addition of special chemicalmembranes on the gate insulator of a basic-FET allows the realization ofISFETs (Ion sensitive FETs) for different ions (Ca²⁺, Na⁺, K⁺, . . . )or ENFETs (enzyme sensitive FETs) for other metabolites (glucose,lactose, . . . ). Typical sensitivities of ISFETs are 50 mV/pH and 30mV/pNa(pK) for FETs made with Si₃N₄ and Al₂O₃ gate insulators,respectively. ENFETs for glucose currently have slow response times (3-5min) and go into saturation. Sensors for the neurotransmittersadrenaline and serotonin incorporate the use of Au nanoparticles theyreach sensitivities of 1×10⁻⁶ M and 6×10⁻³ M, respectively. TypicalISFET sensing gate areas are large, typically 400 μm×20 μm, to maximizetheir sensitivity.

Microelectrodes can be used to measure potential differences between theinside of the Microbottle and a reference electrode that will be eitherin a different container or on the other side of the membrane cap. Theelectrode material is very important in this type of application.Corrosion must be taken into account and avoided in order to makeaccurate, repeatable measurements. Possible electrode materials includegold and silver (Ag—AgCl). Since FETs and microelectrodes are fabricatedusing standard microelectronic processes, integrating them with standardCMOS preamplifier and signal processing logic is feasible. While theprocesses are similar, the materials needed to form the FET gates andthe microelectrodes are not standard to CMOS processing. Therefore,careful consideration should be made to the integration of thesepossibly incompatible materials together in a single “chip”. Also properpassivation materials can be utilized to prevent ionic contaminationfrom the cellular solution in CMOS devices. The integration of thesespecialized sensors with the CMOS circuitry may increase the performanceof the Microbottle and enable the coupling of various Microbottles toform programmable multicellular units.

Microfluidics: The micro fluidics layer allows the control of fluids onboth sides of the membrane cap. The microfluidics layer can be eitheranodic or fusion bonded to silicon layer containing sensing and controlelements. Some embodiments are shown and disclosed in the specificationusing liquid PDMS BioMEMS fabrication technology. In one embodiment,alternatively, a microfluidics layer includes channels and siliconcontainers with submicron holes. The channels and containers are etchedinto the substrate by Reactive Ion Etching (RIE) of silicon nitride maskand a non-isotropic KOH etch. The silicon nitride mask is typically lessthan one micron thick and can also be used for membrane structureswithout additional fabrication steps. The pyramidal container istherefore spanned with a silicon nitride membrane. The submicron hole inthe membrane can be fabricated by focused ion beam.

The channels can be connected to tubing leading to external valves andpumps. Active and passive valves can be incorporated as well. A passivevalve acts as flow restrictor and includes a metal and a polyamidemembrane with holes in different positions. An active valve includes anelectrostatic- or pneumatic-deflectable membrane on a segmented hole.

Device Fabrication: The etching of channels and insulation layers, thefabrication of the boron-doped diamond microelectrodes, and thenanoscale machining such as the drilling of the holes for theMicrobottle are developed accordingly for the present invention. Theoxygen sensor, MEMS microfabrication, thermometer deposition andmicromachined infrared detectors are also developed and utilized.Membrane Cap: In one embodiment, the silicon container is spanned byeither a biological membrane harvested from a cell (approximately 10 μm)or by a synthetic membrane assembled on the microfluidics layer. Themembrane forms a seal not only acting as chemical barrier but alsopreventing leakage of currents from the Microbottle electrode to thereference electrode. The resistance is critical for determining theelectrical background noise from which the channel currents need to beseparated. In a typical patch clamp experiment, where the membrane isattached to a glass pipette the resistance is typically gigaohms. Activeelements like voltage sensitive channel are inserted into the membranecap and are either used as sensors or actuators. The voltage sensitivechannels could be switches with an electrode configuration on the rim ofthe hole.Synthetic Membrane: One of the common applications of lipid bilayers hasbeen to study ion channel transport characteristics. Several issues areimportant in the application of bilayers as biosensors. The mostcritical physical properties are membrane uniformity and membranestability and the present invention is capable of addressing theseissues. Lipid bilayers have been deposited on solid platinum, gold andsilicon surfaces. There are several examples of bilayer spanningapplications such as across micro-machined polyimide 40 μm diameterapertures. The microbottle may be temporarily filled with a supportmaterial, while the bilayer is formed. An enzymatic cleavage strategycan be utilized to remove the gels through the fluid channel accessports of the microbottle. The individual molecules forming ion channelscan be inserted in artificial lipid bilayers. Far more complex systems,which employ high-gain biological amplification and therefore thedetection of single molecules, e.g., hormone receptor systems, may alsobe employed.Natural Membrane: The Microbottle according to the present inventionallows fluids to be sucked through one of the holes in the siliconcontainer. By sucking and manipulating a cell onto the top of thesilicon container the cell membrane can be punched open allowing accessto the intracellular space. Natural membrane can be extracted fromvarious different cells by rupturing the cell membrane.Cellular Cap: The Microbottle of the present invention allows fluids tobe sucked through at least one hole in the silicon container. By suckingand manipulating a cell onto the top of the silicon container the cellmembrane can be punched open allowing fluidic and electrical access tothe intracellular space. The extracellular space can also be monitoredthrough a second fluidics layer encapsulation the cell. The cell is nowan active elements; the intra- and extra cellular space is monitored andcontrolled through the silicon base layer and the microfluidics layers.Such an embodiment is shown in FIG. 5. The immediate spin off thistechnology is a new measurement technique with unsurpassed possibilitiessuperseding conventional patch clamp techniques. Small pore diameterscan only be obtained in glass micro-pipettes if the cone angle is verysmall and the pipette resistance correspondingly high, the RC noisegenerated by this resistance in conjunction with the distributed pipettecapacitance limits the bandwidth of voltage recordings dramatically. Thelong conical shape of the glass micropipette also restricts theselective perfusion of the intracellular space. In contrast, theMicrobottles does not impose geometrical constrictions and allows thecontrolled fabrication of ultra small pores beyond the capabilities ofglass pipettes. The low access resistance of the silicon micropipetteused with small membrane patches brings the potential of voltageclamping in the megahertz frequency domain. The present invention alsoallows temporal resolution of a variety of important electrogenic eventssuch as ion-binding reactions and fast conformational changes associatedwith transport function.

In tiny patches the probability of the appearance of any othercharge-translocation processes is reduced in proportion to the patcharea. Furthermore the formation of a reliable seal becomes increasinglymore difficult as the size of the pipette tip is increased. The wideplanar rim of the silicon chip utilized in the present invention isexpected to reduce the shunt resistance, leads to a greater stabilityand a significantly higher success rate in a patch clamp process. Ingeneral the stability of those small patches is expected to beenormously high with a seal resistance of several hundred gigaohmsallowing long-term recordings. The microbottle would not only simplifyand overcome the limitations of patch-clamp techniques but also movetowards integrating biologically active components into electroniccircuits on silicon wafers.

A plurality of Microbottles of the present invention can be arranged incomplex array structures allowing to readout and stimulation of cellularnetworks. The cellular network would be fabricated with a techniquecalled soft lithography. According to one embodiment of the presentinvention, in the first step pits and connecting channels are etchedinto silicon substrates. After etching, the channels and pits are coatedwith an adhesive protein (polylysine), which promotes cell adhesion andcell growth. After coating, neural or cardiac cells are platted onto thesilicon substrate the cells adhere in the pits and form dentrides alongthe channels connecting to neighboring cells. The chip with thepatterned cellular networks is “flip chipped” to the silicon wafercontaining the Microbottles. Since the Microbottles probe and controlthe intracellular space, a well-defined cell-silicon coupling can berealized. Conventional techniques like microelectrode arrays or cellpotential FETS (CPFETs) suffer from poor coupling and therefore reducedsignal amplitudes. Recordings are generally on the order of 10-200 μVcompared to 80 mV in patch clamp techniques.

Example 5 Picocalorimeter and Bioreactor

In one aspect, the present invention relates to a device for measuringresponse of at least one cell 203 to a medium, the response of at leastone cell 203 to a medium being characterized by a reaction time. In oneembodiment as shown in FIGS. 2A and 2B, a device 200 includes a membrane206 having a first surface 251, an opposite second surface 253 and edges255, a side substrate 212 having an inside surface 261, an oppositeoutside surface 263, a top surface 265 and bottom surface 267, whereinthe inside surface 261 of the side substrate 212 and the first surface251 of the membrane 206 define a sample well 269 in communication withthe ambient air and for receiving the at least one cell 203 such thatthe membrane 206 is underneath the at least one cell 203, a sensor 205positioned underneath the second surface 253 of the membrane 206, and aninlet 210 in fluid communication with the sample well 269. A medium (notshown) is introduced into the sample well 269 through the inlet 210 toform a droplet 209 that surrounds the cell 203, and the sensor 205measures the response of the cell 203 to the medium at a time periodshorter than the reaction time. The sensor 205 is in contact with thesecond surface 253 of the membrane 206. The response of the cell 203 tothe medium, which may contain at least one agent or stimuli, depends onthe characteristic of the cell 203 as well as the properties of theagent such as the type or class of the agent. Therefore, among otherthings, one application of the device 200, and other device 200 s andmethods of the present invention, is to detect the agent from theresponse of the cell 203 to the medium having the agent, which uses thecell 203 as canary.

The device 200 also includes a biocompatible coating layer (not shown)applied to the first surface 251 of the membrane 206. The membrane 206comprises a material with sufficiently low thermal conductivity to yielda high degree of thermal isolation between the center of the membrane206 and the edges. For examples, the membrane 206 may comprise adielectric material. The membrane 206 may also comprise a siliconnitride membrane 206. Moreover, the membrane 206 is at least partiallytransparent so that the response of the cell 203 can be opticallydetected through an optical sensor.

The inside surface 261 of the side substrate 212 makes contact with thefirst surface 251 of the membrane 206 to define the sample well 269,wherein the side substrate 212 is thermally isolated from the membrane206. A hydrophobic layer 204 can be applied to the inside surface 261 ofthe side substrate 212.

In one embodiment as best shown in FIG. 2A, the inside surface 261 ofthe side substrate 212 has a slope defined by an angle α such thatcross-sectionally the bottom surface 267 of the side substrate 212 iswider than the top surface 265 of the side substrate 212. The sidesubstrate 212 comprises a material with sufficiently high thermalconductivity such that the side substrate 212 functions as a heat sinkfor the membrane 206. For examples, the side substrate 212 may comprisea semiconductor material such as silicon.

The sensor 205 can be any type of sensor as defined and discussed above.As an example shown in FIGS. 2A and 2B, the sensor 205 can be a thermaldetector.

The device 200 further includes an actuator 202 that is mechanicallycoupled to the inlet 210. The inlet 210 has a main portion 271 and anend portion 273 in fluid communication with the main portion 271. Theend portion 273 is movable between a first position 273 a that isdistant from the cell 203 and a second position 273 b that is proximateto the cell 203. When a medium is to be introduced into the sample well269, the actuator 202 causes the end portion 273 to move away from thefirst position 273 a to the second position 273 b or a positiontherebetween the first position 273 a and the second position 273 b fordelivering the medium to the sample well 269 to form a droplet 209 toisolate the cell 203. After a medium is introduced into the sample well269 and droplet 209 is formed, the actuator 202 can cause the endportion 273 to move toward to the first position 273 a from the secondposition 273 b or a position therebetween the first position 273 a andthe second position 273 b for keeping the end portion 273 away from thedroplet 209 isolating the cell 203.

The device 200 further has a control 201 positioned inside the mainportion 271 of the inlet 210 for controlling the flow of the medium.Additional controls 201 can be positioned at branches in fluidcommunication with main portion 271, as best shown in FIG. 2B, such thatthe content of the medium can be adjusted as needed.

The device 200 also has an outlet 211 in fluid communication with thesample well 269 for introducing medium away from the sample well 269. Anactuator 252 mechanically coupled to the outlet 211. The outlet 211 hasa main portion 281 and an end portion 283 in fluid communication withthe main portion 281. The end portion 283 is movable between a firstposition 283 b that is proximate to the cell 203 and a second position283 a that is distant from the cell 203. When a medium is to beintroduced away from the sample well 269, the actuator 252 causes theend portion 283 to move away from the first position 283 b to the secondposition 283 a or a position therebetween the first position 283 b andthe second position 283 a for introducing the medium away from thesample well 269. After a medium is introduced away from the sample well269, the actuator 252 may cause the end portion 283 to move back to thefirst position 283 b from a position therebetween the first position 283b and the second position 283 a. The device 200 further has a control251 positioned inside the main portion 281 of the outlet 211 forcontrolling the flow of the medium. Additional controls 251 can bepositioned at branches in fluid communication with main portion 281, asbest shown in FIG. 2B, such that the content of the medium can beadjusted or controlled as needed.

Thus, a device according to the present invention is shown to be able tomeasure the energy generation and consumption of a single or multiplecells. In some embodiments, such a device is termed as aPicocalorimeter. As shown in FIGS. 2A and 2B, in operation, device 200uses a membrane 206 in combination with a sensor 205 to measuremeasurable quantities related to the status of a single or multiplecells such as the basal energy generated by a cell 203 in a droplet ofculture media 209. The droplet of cell culture media is confined on themembrane 206 by a hydrophobic coating 204. The temperature differencebetween the membrane and the substrate 212 is measured with sensor 205,which can be a differential sensor and may be coupled to additionalcomponents such as an amplifier 213 through leads 214. The content ofthe droplet 209 can be exchanged to maintain cell viability using inlet210 that can have an optional branch structure formed by a number ofinlet lines 217 as shown. The content of the inlet structure can bevaries using valves 201, which are driven by controller 208. Foranalysis of the content in the outflow through outlet structure, thefluid can be switched by valves 251 to have various analyzing structuresformed by a number of outlet lines 218. The inlet structure 210 can bepositioned by actuator 202 to inject fluid into the droplet and toretract from the droplet 209 to maintain thermal insulation,respectively. The outlet structure 211 can be positioned by actuator 252to withdraw fluid from the droplet 209 and to retract from the droplet209 to maintain thermal insulation, respectively. During the measurementinterval the inlet and the outlet structure are retracted. Additionalelectrodes 207 can be used to monitor various metabolites in the droplet209 in order to detect, for example, metabolic pathway switching.Controller 208 allows the analytes in the media to be changed dependingon the status of the metabolic network in order to override internalcellular control.

Moreover, in another embodiment (not shown), MEMS and microfluidictechnologies are utilized to provide a flow-through system in which theheat production of a small number of cells may be monitored before andafter the cell stream merges with the injected flow of medium containingagent such as toxin. Micropipes can be electrically actuated by piezobimorphs so that they can be separated from the droplet to thermallyinsulate the cell on the membrane in the thermal measurement interval.Beside heat generation, oxygen, pH and Redox potential sensors can beintegrated on chip as well as advanced readout and control electronics.

In another aspect, the present invention relates to a device formeasuring at least one of cellular physiological activities of at leastone cell or cells, where each of the cellular physiological activitiescan be characterized by a reaction time. In one embodiment as shown inFIGS. 25A and 25B, the device 2500 includes a membrane 2505 having afirst surface 2551, an opposite second surface 2553 and edges 2555, aside substrate 2504 thermally isolated from the membrane 2505 and havingan inside surface 2561, an opposite outside surface 2563, a top surface2565 and bottom surface 2567, wherein the inside surface 2561 of theside substrate 2504 cooperates with the first surface 2551 of themembrane 2505 to define a sample well 2501 in communication with theambient air and for receiving the at least one cell or cells 2503 suchthat the membrane 2505 is underneath the at least one cell 2503, and asensor 2506 positioned underneath the second surface 2553 of themembrane 2505 for measuring at least one of cellular physiologicalactivities of the at least one cell 2503. The membrane 2505 and thesensor 2506 are arranged such that at least one of cellularphysiological activities of the at least one cell 2503 is measured at atime period shorter than the reaction time. Note that among otherthings, one advantage of the device 2500 is that it allows operation inair, which makes the utilization of living cells possible and alsoeliminates many disadvantages related to the requirement of operation invacuum by the prior art such as cost, inconvenience, low reliability,etc.

As discussed above and below related to the present invention, amongother things, it can be done by limiting the dimensions of the device,positioning the sensor(s) proximate to the cell(s), and/or choosingcellular physiological activities to be measured such that quantitiesrelated to the status of a cell, being a response to an agent or acellular physiological activity, can be obtained quickly before acorresponding reaction time such as a diffusion time. Thus, one uniqueaspect of the present invention is that small is faster and better. Itwill be appreciated, however, that in addition to a reaction time thatcharacterizes a response to an agent or a cellular physiologicalactivity of a cell, other quantities may be considered as well. Forexamples, in one embodiment, the device 2500 can be utilized to detectsignals corresponding to the amount of heat generated by cells as afunction of time or intervention. This can either be as a measurement ofpower, or total energy change. The characteristics of the media that arein fluid communication with the cells include the thermal conductivityand the heat capacity. These may combine to give the response time andresponse amplitude for the system when cells within the sample wellproduce heat. Device 2500, like other embodiments of the presentinvention, allows one to obtain proper signals at proper places within aproper time period.

The device 2500 also may include a biocompatible coating layer (notshown) 115 applied to the first surface 2551 of the membrane 2505 forbetter housing the cells 2503. The membrane 2505 comprises a materialwith sufficiently low thermal conductivity to yield a high degree ofthermal isolation between the center of the membrane 2505 and the edges2555. For examples, the membrane 2505 may comprise a dielectricmaterial. The membrane 2505 may also comprise a silicon nitride membrane2505. Moreover, the membrane 2505 is at least partially transparent sothat the response of the cell 2503 can be optically detected. Thedimensions of the membrane 2505 can be chosen such that measurements canbe performed at a desired time period that is shorter than acorresponding reaction time. For examples, in one embodiment as shown inFIGS. 25A and 25B, the thickness of the membrane 2505 is in the range of0.1 to 1.5 μm, and the size of the membrane 2505 is in the range of 0.1to 25 mm². In a particular example, the thickness of the membrane 2505is chosen as about 0.6 μm, and the size of the membrane 2505 is chosenas about 1 mm².

The inside surface 2561 of the side substrate 2504 makes contact withthe edges 2555 of the membrane 2505 to define the sample well 2501,wherein the side substrate 2504 is thermally isolated from the membrane2505. A hydrophobic layer (not shown) can be applied to the insidesurface 2561 of the side substrate 2504.

In one embodiment, the inside surface 2561 of the side substrate 2504has a slope with an angle β such that cross-sectionally the bottomsurface 2567 of the side substrate 2504 is wider than the top surface2565 of the side substrate 2504. The side substrate 2504 comprises amaterial with sufficiently high thermal conductivity such that the sidesubstrate 2504 functions as a heat sink for the membrane 2505. Forexamples, the side substrate 2504 may comprise a semiconductor materialsuch as silicon.

Again, the sensor 2506 can be any type of sensor 2506 as defined below.As an example, the sensor 2506 can be a thermal detector. In oneembodiment as shown in FIGS. 25A and 25B, the thermal detector 2506comprises a thermometer, wherein the thermometer comprises a thermopilehaving a first polarity of junction positioned underneath and in contactwith the second surface 2553 of the membrane 2505 and a second polarityof junction thermally coupled to the side substrate 2504. The thermopileincludes a series of thermocouples, wherein an emf measured at the leadsof the thermopile is proportional to the temperature difference betweenthe membrane 2505 and the side substrate 2504. Alternatively, thethermometer can be a resistive thermometer having a series of resistorselectrically coupled to each other. Additionally, other sensor(s) can beutilized to cooperate with the thermal sensor 2506. For instance, anoptical sensor (not shown) can be utilized to optically detect thestatus of the cells 2503 through an at least partially transparent area2573 of the membrane 2505.

Thus, a Picocalorimeter according to one embodiment of the presentinvention combines the highly complex and evolved sensing architectureof cellular systems and a new micro-machined silicon transducer devicecapable of detecting minute heat changes arising from changes in themetabolism of a single cell with a response time of a few milliseconds.The Picocalorimeter, with a sensitivity of 1-50 pW, results in animprovement of more than four orders of magnitude and hence achievesingle cell sensitivity. The measured specifications of micromachinedcalorimeters, and calculated values for optimized shown in the followingtable.

Measured: Calculated: optimized Specification existing prototype deviceDetector area 1 mm² [0.3. mm]² Responsivity 110 volts/watt 1365volt/watt Noise 110 nV/{square root over (Hz)} 80 nV/{square root over(Hz)} Time constant 50 msec 15 msec Minimum detectable 1000picowatt/{square root over (Hz)} 59 picowatt/{square root over (Hz)}power Energy Sensitivity, 130 picojoule 7 picojoule τ = 10 s

The Picocalorimeter can be fabricated by employing micro-machiningtechniques. As discussed above, a Picocalorimeter includes a siliconnitride (SiN) membrane, thermally insulated from the silicon wafer, andone or more thermometers in the center of the membrane. The thermometeris typically a series of resistors or thermocouples forming athermopile. The heat quantity evolved or absorbed is equal orproportional to the product between temperature change and the heatcapacity of the calorimetric vessel and its contents. Since the membranedimensions are small and silicon nitride has an extremely low-thermalconductivity and heat capacity, the device is intrinsically sensitive.By minimizing the total thermal conductance, a small quantity of heattransferred to or from the sample results in a large, measurabletemperature rise. For the optimized device, about 20 μW of power willraise the Picocalorimeter temperature by 1 K. The mK sensitivity of thethermometer gives pW resolution. This may be achieved by employingmicromachining fabrication to produce a rigid membrane only 0.6 μm thickor less.

The contents of the Picocalorimeter can be single or multiple cellsgrown or placed onto the SiN membrane and a drop of liquid surroundingthe cell. Several measurements on membranes have shown a high mechanicalstability of the silicon nitride membrane and its ability to support aliquid droplet, and growing cells directly on the membrane. The liquidcontains all required nutrients and may be periodically exchanged by twonanoliter injectors (not shown) between measurement intervals. Twonanoliter injectors are mounted on computer-controlledmicro-positioners, which can also be used to position the cells on theSiN membrane or to introduce agents that alter the metabolism of thecell. The following table shows the metabolic activity of various celllines of interest.

Cell types Power/heat output Reference Human T-lymphoma cell 12.2pW/cell P. Backman, 1991 Human melanoma cells   80 pW/cell M.Gorman-Nordmark et. al 1984 Rat white adipocytes   40 pW/cell P.Nilsson-Eble et. al 1985 3T3 mouse fibroblasts   17 pW/cell P. Lonnbroet. al 1990 Rat hepatocytes  329 pW/cell L. Nassberger et. al 1986 Humankeratinocytes   40 pW/cell U. Reichert et. al 1986

The metabolically complex liver cell generates 350 pW/cell and may be anideal sample to demonstrate the applicability of the Picocalorimeter.Such a single liver hepatocyte could be monitored with a 5:1 signal tonoise ratio. The sensitivity of the Picocalorimeter can be utilized tomeasure the dose-response relationship between a beta-adrenergic agonistand heat production in hepatocytes. The effects of Dinitrophenol, whichuncouples heat production from oxygen consumption, on heat productioncan then be examined, which in turn can be used to optimize thePicocalorimeter and determine its sensitivity and the response times tofluctuations in heat production.

The microfluidic components and controls discussed else where in thespecification may be adapted, incorporated and enhanced to allow forthermal isolation of each cell environment. The Picocalorimeter with theliving cell may be hermetically sealed with a cap to protect the cellenvironment from contamination. Supply and waste microchannels mayconnect to micropipettes that can be moved in and out of the liquid dropsurrounding the cell. Actuation of the micropipettes can be accomplishedby either deflection of piezo bimorphs connected to the micropipettes,or deflection of the sealing membrane by the piezoelectric filamentarray also used to actuate the pumps and valves.

In yet another aspect, the present invention relates to a device formeasuring at least one of cellular physiological activities of at leastone cell, where each of the cellular physiological activities can becharacterized by a reaction time. In particular, the device can beutilized to measure the energy generation and consumption of a single ormultiple cells. In one embodiment as shown in FIGS. 6(A)-(D), a device600 includes a membrane 606 having a first surface 651, an oppositesecond surface 653 and a thickness, wherein the membrane 606 has asensing area 659 for receiving the at least one cell 611 such that themembrane 606 is underneath the at least one cell 611. The device 600further includes a substrate 602 positioned opposite to the membrane 606and having an inside surface 661 and an opposite outside surface 663,wherein the inside surface 661 of the substrate 602 cooperates with thefirst surface 651 of the membrane 606 to define passage 670therebetween. The substrate 602 has a first flexible portion 665 locatedat one side of the sensing area 659 of the membrane 606 and a secondflexible portion 667 located at another side of the sensing area 659. Asensor 607 is positioned underneath the sensing area 659 of the membrane606 for measuring at least one of cellular physiological activities ofthe cell 611. The membrane 606 and the sensor 607 are arranged such thatat least one of cellular physiological activities of the cell 611 ismeasured at a time period shorter than the reaction time.

The device 600 may also include a biocompatible coating layer (notshown) applied to the first surface 651 of the membrane 606. Themembrane 606 comprises a material with sufficiently low thermalconductivity to yield a high degree of thermal isolation between thecenter or sensing area 659 of the membrane 606 and the edges. Forexamples, the membrane 606 may comprise a dielectric material. Themembrane 606 may also comprise a silicon nitride membrane 606. Moreover,the membrane 606 is at least partially transparent so that the statusand/or response of the cell 611 can be optically detected. Thedimensions of the membrane 606 can be chosen to meet different needs.

The first flexible portion 665 of the substrate 602 has a firstdiaphragm that is actionable by a force. When a force is applied to thefirst diaphragm, the first diaphragm moves along the direction of theforce. As an example, the first diaphragm can be a PDMS membrane.

The second flexible portion 667 of the substrate 602 has a seconddiaphragm that is actionable by a force. When a force is applied to thesecond diaphragm, the second diaphragm moves along the direction of theforce. As an example, the second diaphragm can be a PDMS membrane. Inthe embodiment as shown in FIGS. 6(A)-(D), the substrate 602 is a PDMSmembrane.

The first diaphragm and the second diaphragm can be utilized jointly orindividually in operation. For examples, when a first force is appliedto the first diaphragm towards the outside surface 663 of the substrate602, the first diaphragm moves along the direction of the force to reachto the first surface 651 of the membrane 606, and when a second force isapplied to the second diaphragm towards the outside surface 663 of thesubstrate 602, the second diaphragm moves along the direction of theforce to reach to the first surface 651 of the membrane 606, thereby toform an isolated region therebetween. In the embodiment as shown inFIGS. 6(A)-(D), the first flexible portion 665 and the second flexibleportion 667, when pushed by air pressure through inlets 601, form anenclosed measurement volume 617 containing the cell 611 therein andisolating the cell 611 from communication with fluid outside themeasurement volume 617. Note that the first diaphragm and the seconddiaphragm can be separate elements, or integral parts of a ring-shapeddiaphragm that substantially encircles the measurement volume 617, asshown in FIG. 6.

Conversely, when at least one of the first force and the second force iswithdrawn, a corresponding one of the first diaphragm and the seconddiaphragm moves away from the first surface 651 of the membrane 606,thereby to allow the isolated region inside measurement volume 617 influid communication at least partially with the passage 670.

Thus, as shown in FIGS. 6(A)-(D), device 600 can be utilized to measurethe energy generation and consumption of a single or multiple cells. Thedevice 600 uses a first membrane 606 that is sufficiently thin with asensor 607 to measure the basal energy generated by a cell 611 in ameasurement volume 617 thermally isolated from the surrounding using agas/air filled space bounded by a second membrane 602 that issufficiently thin. By pressurizing the gas filled chamber 613 throughthe inlets 601, the chamber 613 can be expanded to isolate the liquidmedia surrounding the cell 611 in the passage 670 to form a droplet,which has inlet 615 and outlet 616 for supplying a stream of fresh mediaand draining spend media from the measurement volume 617, respectively.Valves 610 can be utilized to allow the control of the inflow andoutflow of the stream through the passage 670. On top of the measurementvolume 617, there is a gas filled chamber 604, which is stiffened bybridges 612 or is pressurized to avoid a collapse or deformation of themeasurement volume 617 when the membrane 602 is inflated during themeasurement cycle. Additional electrodes 608 can be used to monitorvarious metabolites in the droplet in order to detect, for example,metabolic pathway switching. The electrodes 608 can be coupled throughthe lead 614 to a sensing unit (not shown). It will be appreciated thatmeans other than pressurized gas can be used to move the first flexibleportion 665 and the second flexible portion 667, which could also be anyother low-thermal conductivity barrier or object that may bemechanically placed around the cell with a piezoelectric or othermechanical actuator and the like.

Example 6 Signal Extraction and Discrimination

It will be appreciated that practicing the present invention ofteninvolves apparatuses or devices that have biological, electronic andmicrofluidic components interfacing each other and interacting together.To build these devices, a design theory is developed that is supportedby an integrated modeling paradigm (language) that allows the modeling,analysis, simulation, and synthesis of these hybrid systems. Theinformation or INFO component of the present invention develops thismodeling language, and selects and/or builds analysis and synthesistools for the present invention. One of them is a Bio-Micro CAD toolthat can be used by biologists, biochemists, and diagnosticians toproduce biocontrollers.

Currently, there is no integrated effort that would address all of theneeds of a bio-silicon hybrid device. Silicon behavioral (analog ordigital circuit) simulators exist, and there are efforts for engineeringbiological processes using (circuit) simulation techniques, but there isno integrated modeling and analysis framework that would combine thetwo. In one aspect, the present invention provides an integratedbio-silicon-hybrid system design environment to meet the need.

As schematically shown in FIG. 8, a design environment 800 according tothe present invention has multiple customized interfaces 801communicating with users including microbiologists, hardware/sensorengineers, and diagnostic experts. The design environment 800 has amicrocontroller 810 that, among other things, contains models 802-805operated therein, receives inputs from the interfaces 801 and drivessystem generators 806, 808. System models include biological models 802that capture cellular metabolic cycles including cellular products,physical models 803 that capture sensor configurations, digitalprocessors, fluid processing hardware, and other devices on the chip,cell diagnosis models 804 that capture the differential diagnosisprocedure including measurement parameters, decision logic based onmeasured/computed parameters, and physical actions to change cellularenvironmental parameters, control strategy models 805 that define how toachieve the cellular environmental parameter changes using the hardwaredefined in the physical models 802. Models can be added, deleted,edited, modified, tested, operated, used, saved, and upgraded, amongother things. Moreover, the design environment 800 has one more systemgenerators for various tasks. For examples, system generator 806 createssimulations of the procedures and physical structures as defined by anyor any combination of models 801-805 and generates simulationconfiguration data 812 to drive a biological simulator 813, in whichoffline simulations execute these models 807, and produce data that canbe used to optimize corresponding models 801-805. System generator 808converts the models 801-805 into executable code 811 that contains thesoftware and hardware configuration information, and runs on thephysical device 809 to perform cell diagnostics in theapplication-specific bio-microcontroller 814 accordingly. Moredescriptions about what the design environment 800 can do are givenbelow.

In one aspect, the present invention relates to a method fordiscriminating an agent. To do so, a process for agent classificationneeds to be defined. In one embodiment, overall agent classification canbe implemented in the design environment 800 by successive refinement ofdiagnostic hypotheses. The steps in this process can be defined using adiagnosis tree having a plurality of branches. At each branch of thetree, a context-sensitive experiment is conducted and data acquired andanalyzed. The context is a result of all prior experiments anddecisions. At each experiment/assay, the following steps are performed:

Setting assay conditions, including: (a) Selecting the type and quantityof cells to be exposed; (b) Modifying the ‘set-points’ of the cell,(e.g., changing pH to make the cell metabolism more sensitive to aparticular protein, etc.); (c) applying the unknown agent to the cellswith a user-specified profile;

Acquiring data from the sensors, and processing it into “features”.Features represent processed information from the raw signal, convertinga time sequence of raw A/D sample counts into a small number ofparameters. Arbitrarily complex feature extraction algorithms can bedefined by connecting software modules from a library of signal analysisfunctions (e.g., slope, frequency analysis, parametric modeling, etc.)in a Lab View-like environment; and

Applying a discrimination function to evaluate the features, dividinginto classes of responses. The classification assurance will be assessedand used to select the next branch of the decision tree. Discriminationfunctions can be implemented to form a library of techniques (PrincipalComponent/Factor Analysis, Statistical Clustering/Maximum LikelihoodEstimation, Parametric Model/System Identification, Neural Networks,User-Defined, etc.).

In operation, care should be taken to integrate control and diagnosis toconserve valuable resources (limited number of cells, limited quantityof reagents), and modify cell conditions to enhance sensitivity of thebiological systems to improve quality (e.g., to increase probability ofdetect, to reduce false alarm rate, etc.).

Defining the diagnosis tree is an experiment- and data-intensive,iterative process. As shown in FIG. 8, the design environment 800 iscapable of supporting programming of the system by a plurality of users801. A Model-Integrated Computing (“MIC”) approach is utilized to designand implement a Domain-Specific language for agent classification. TheMIC approach has proven successful at a wide variety of embedded systemsand diagnostic domains.

Discrimination can be achieved in several ways according to the presentinvention. Referring now to FIG. 22, a differential discriminationprocess is shown. In step 1, a series of incremental refinementprocedures 2201 are defined as a decision tree. Starting with a broadsweep assay, and using the results of a discrimination process, theagent is identified as being either viral or neural. Moreover, classesand subclasses of neural responses are identified by choosing a newassay to further refine decisions, which can be considered as branchesand successive branches of the decision tree. Refinement proceeds untilsufficient confidence and discrimination is achieved. In Step 2,experimental conditions and control setting parameters 2202, such ascell type and quantity to expose to the unknown agent, cell bathcomposition, or the like, are defined. In Step 3, the individual entries2203 in a first database, where is shown as Data Point Library, are usedto define a detailed discrimination process that involves specificmeasurements 2204 such as fast Fourier transforms (“FFT”), summations,differences, and feature extraction, during which a feature extractionalgorithm is graphically defined in a second database 2205, where isshown as Feature Extraction Library, that contain common signalprocessing primitives to produce the resulting feature 2206 that isquantified. At step 4, discrimination functions or discriminators aredefined by using several features 2207 to indicate differentialdiagnostics paths to choose, and/or obtained from a third database 2208,where is shown as Discrimination Functions Library, and a historicaldata base 2209 that contains common discrimination functions andprovides a historical record available for use in discrimination andalgorithm verification. As a result, one or more discriminationfunctions, i.e. classification information about the agent, aregenerated, which can be used to select the next branch of the diagnosistree. These discriminators can also be used to discriminate or classifythe agent from the measured signals corresponding to the agent. Theprocess can be repeated until the agent is discriminated.

Accordingly, as shown in FIG. 22 and discussed above, one method 2200for discriminating an agent according to one embodiment of the presentinvention includes the steps of (a) constructing a decision tree 2251having a plurality of branches 2253, 2255, each branch corresponding toat least one defined action, wherein each branch includes a plurality ofsuccessive branches 2257, 2259, each successive branch corresponding toat least one defined action, (b) providing a conditioned environment2202 sensitive to the agent, (c) obtaining data from response of theagent to the conditioned environment at 2203, (d) extracting featuresfrom the obtained data at 2204, (e) selecting a branch from the decisiontree corresponding to the features at 2206, (f) performing on thefeatures at least one defined action corresponding to the branch, and(g) producing a classification of the agent at 2207. Some of the abovesteps can be iteratively repeated until the agent is discriminated.

In doing so, as shown in FIG. 22, at the start, a decision is maderegarding the choice of logic for successive refinement of agentclassification, where it can be chosen as logic for classification of aNeuro agent at 2255, or logic for classification of a Viral agent at2253. The agent may include a chemical agent, a non-chemical agent, abiological agent, or a non-biological agent. Examples of the agent canbe found above.

Different signal classification algorithms can be utilized to classifyan agent. In one embodiment as shown in FIG. 23, for the process 2351shown at the upper half, sets of available measurements 2301, where eachmeasurement represents a parameter related to the status of the agent,for example, the entry of a toxin to a conditioned environment havingcells may cause temporal response measured as T, are chosen and used asinput data to the feature extraction process 2302 that graphicallydefine algorithms to extract properties from the raw cellularmeasurements. From these data, feature sets 2303 are computed fromsolving a set of first order differential equations although secondorder, or high order differential equations may also be used. Featuresets 2303 are then analyzed at 2304 by classification algorithmsincluding principal component analysis and other linear/nonlinearclassifiers to separate the features into a feature space 2305. Space2305 is an example of a poorly classified decision, where featuredistributions overlap to each other, based upon a particular featureextraction process 2302. A similar process 2353 as shown in the lowerhalf of FIG. 23, however, produces a feature space 2306 that representsa good classification of three regions from a different extractionprocess. One difference is that process 2353 uses a set of second orderdifferential equations at step 2312 as discussed in more detail below.

Indeed, an important phase of signal classification is the initialfeature extraction. Feature extraction can take the form of simplemathematical operations on signals (add/subtract, computeslope/area-under-curve) or can incorporate metabolic or otherphysiological information such as intracellular or intercellularsignaling activity via parameter matching to biological models. Designof the feature extraction algorithms is an iterative process aspartially shown in FIG. 23. As shown in the upper half of FIG. 23, theinitial attempt 2351 selects a set of sensor inputs (temperature,oxygen, pH, etc.). A first-order differential equation model ofmetabolic pathways is used to extract features from T, pH, and NADPHamongst other algorithms. A Principal-Component-Analysis (PCA)/Clusterseparation reveals that the classes are only separable with a 20%confidence level.

In the lower half of FIG. 23, a refinement of the feature extractionshown as process 2353 changes the biological model to 2nd order and addsa new model as a feature (O—). Successive PCA shows that the classes arenow separable with a 90% confidence level. Among other things, thefeature extraction primitives include (1) Standard mathematical/DSPfunctions, (2) Model Parameter Identification for 1st, 2nd, and 3rdorder rate equations, (3) Mean transit time and Impulse response models,and (4) Kinetics of mass/heat diffusion. In addition, generic ‘shells’will be available to perform user-defined analysis.

Classified data are stored in a database for further use. However, whenbuilding the experimental classification database in an unsupervisedmode, the input to the algorithms are unlabelled examples. Unsupervisedclassification algorithms are used to discover natural structures in thedata and can provide valuable insight into the problem and guide thedevelopment of classification system. As described above, the designenvironment 800 can be used for a wide range of applications. On one endof the spectrum, it can be used to design decision trees that are basedentirely on deep physiological knowledge. In this scenario, the numberof features at each decision node would be relatively limited andassignment to one class or the other would be made on the goodness offit between data and model. On the other end of the spectrum it can beused to design classification systems even if very little is known aboutphysiological principles. In this scenario, the number of features wouldbe large, the system provided with labeled examples, and it would simplycompute decision boundaries in the feature space.

FIG. 24 illustrates a diagnostics path to develop an assay according toone embodiment of the present invention. Note that this is illustrativeof the definition process and represents a small fraction of a completediagnostics process. The first step is to have a broad spectrum assay.Robust, relatively insensitive cells from cell lines are used to providea long-lived activity detector. The broad assay may separate responsesinto one of several broad classes, discriminated by a Maximum LikelihoodEstimator. FIG. 24 further illustrates the next step for Gram-NegativeBacteria. A new set of engineered cell lines is selected for theirsensitivity to the presence or absence of CD-14 (i.e., endotoxinreceptor), along with instrumentation to measure the anticipatedindicators. The expected response is used to define a set of featureextraction algorithms. In this case, a simple threshold serves as aclassifier. The third step in the path chooses intestinal cells todetermine if a pathogenic enterotoxin is secreted by the gram negativebacteria, a set of sensors, feature extraction algorithms, and a MLEclassifier. Note that there may be additional steps to be performed.

Accordingly, as shown in FIG. 24, diagnosis process 2400 proceeds asfollows. At step 2401, cell lines, sensors, and analysis metadata arechosen and obtained to provide a broad-spectrum activity assay at 2404.Feature extraction algorithms are utilized at 2405 to define how toconvert raw sensor measurements into features at 2406. The featuresresulting from feature extraction are examined by a classifier method at2407, which is selected for its ability to discriminate agent classes.Classification results are used to make a decision of which path toproceed at 2408. For examples, a second set of cell lines, sensors, andanalysis metadata are chosen and obtained to provide a broad-spectrumactivity assay at 2424 and subsequent analysis is repeated to generatenew classification results. For poor discriminations, multiple paths canbe followed at step 2409. Additional subsequent steps that follow thesame procedures, with specific feature extraction and classificationmethods at 2403 (or more) can be repeated until desired results areobtained, for example, when a desired robustness factor is obtained.

While there has been shown various embodiments of the present invention,it is to be understood that certain changes can be made in the form andarrangement of the elements of the system and steps of the methods topractice the present invention as would be known to one skilled in theart without departing from the underlying scope of the invention as isparticularly set forth in the claims. Furthermore, the embodimentsdescribed above are only intended to illustrate the principles of thepresent invention and are not intended to limit the claims to thedisclosed elements.

1. A device for monitoring status of at least one cell, wherein the cellhas a membrane forming a substantially enclosed structure and definingan intracellular space therein, comprising: a. a first substrate havinga first surface and an opposite second surface; b. a second substratesupported by the first substrate, the second substrate having a firstsurface, an opposite second surface, a body portion between the firstsurface and the second surface, a first side surface and an oppositesecond side surface, wherein the body portion defines a first passagetherein between the first side surface and the second side surface, anda first opening on the first surface of the second substrate that is influid communication with the first passage; c. sidewalls positionedabove the first surface of the second substrate; d. a third substratepositioned over the sidewalls and the second substrate and having afirst surface and an opposite second surface, wherein the thirdsubstrate, the sidewalls and the second substrate define a chamber, andwherein the chamber is in fluid communication with a second passagedefined by portions of the sidewalls and the third substrate; e. atleast one sensor positioned in the first passage proximate to the firstopening, wherein the cell is positioned in the chamber and theintracellular space of the cell is in fluid communication with the firstpassage through the first opening of the second substrate; f. at leastone seal element positioned on the second substrate and proximate to thefirst opening, for sealing the cell to the second substrate inoperation; g. a pair of first controls positioned inside the firstpassage for controlling the flow of a medium through the first passage;and h. a second control positioned inside the second passage forcontrolling the flow of a medium through the second passage.
 2. Thedevice of claim 1, wherein the membrane of the cell defines a secondopening through which the intracellular space of the cell is in fluidcommunication with the first passage through the first opening.
 3. Thedevice of claim 2, farther comprising a punching element positionedunderneath the first opening for making the second opening.
 4. Thedevice of claim 3, wherein the punching element comprises anelectroporation device.
 5. The device of claim 2, wherein when a firstmedium is introduced into the first passage and, the intracellular spaceof the cell is in fluid communication with the first passage having thefirst medium, the sensor measures the response of the cell to the firstmedium.
 6. The device of claim 2, wherein when a second medium isintroduced into the chamber through the second passage, and at leastpart of the membrane of the cell is in contact with the second medium inthe chamber, the sensor measures the response of the cell to the secondmedium.
 7. The device of claim 2, wherein when a first medium isintroduced into the first passage, and a second medium is introducedinto the chamber through the second passage, respectively, theintracellular space of the cell is in fluid communication with the firstpassage having the first medium, and at least part of the membrane ofthe cell is in contact with the second medium in the chamber, the sensormeasures the responses of the cell to the first medium and the secondmedium.
 8. The device of claim 1, wherein the first passage is in fluidcommunication with a reservoir of a medium.
 9. The device of claim 1,wherein the second passage is in fluid communication with a reservoir ofa medium.
 10. The device of claim 1, wherein the at least one sealelement is formed and positioned to substantially encircle the firstopening.
 11. The device of claim 10, wherein the at least one sealelement comprises an ohmic element.
 12. The device of claim 1, whereinthe body portion of the second substrate further defines an intersectionportion where the first passage and the first opening are in fluidcommunication, and wherein the intersection portion is at leastpartially formed as a cone shaped portion.
 13. A device for monitoringstatus of a plurality of cells, wherein each cell has a membrane forminga substantially enclosed structure and defining an intracellular spacetherein, comprising: a. a first substrate having a first surface and anopposite second surface; b. a second substrate supported by the firstsubstrate, the second substrate having a first surface, an oppositesecond surface, a body portion between the first surface and the secondsurface, a first side surface and an opposite second side surface,wherein the body portion defines a first passage therein between thefirst side surface and the second side surface and a plurality of firstopenings distributed on and over the first surface of the secondsubstrate, wherein each of the plurality of first openings is in fluidcommunication with the first passage; c. a third substrate positionedover the second substrate, having a first surface and an opposite secondsurface, and spaced apart from the second substrate thereby defining aspace between the second surface of the third substrate and the firstsurface of the second substrate; d. a plurality of sidewalls positionedbetween the second substrate and the third substrate therebypartitioning the space between the second substrate and the thirdsubstrate into a plurality of chambers above the first surface of thesecond substrate such that only one of the first openings distributed onand over the first surface of the second substrate is located betweenthe sidewalls of a corresponding chamber, wherein each chamber is influid communication with at least one neighboring chamber through asecond passage defined over the sidewalls and under the second surfaceof the third substrate; e. a plurality of sensors positioned in thefirst passage, each sensor being proximate to a corresponding one of thefirst openings distributed on and over the first surface of the secondsubstrate, wherein each cell is positioned in a corresponding one of thechambers and the intracellular space of each cell is in fluidcommunication with the first passage through the first opening locatedbetween the sidewalls of a corresponding chamber; f. a plurality of sealelements positioned on the second substrate for sealing a correspondingcell to the second substrate in operation, wherein each seal element isproximate to a corresponding one of the plurality of first openings; g.a plurality of first controls positioned inside the first passage of thesecond substrate, wherein each chamber has a pair of corresponding firstcontrols for controlling flow of the medium through portions of thefirst passage that correspond to that chamber; and h. a plurality ofsecond controls, wherein each second control is positioned inside acorresponding second passage for controlling the flow of a mediumthrough that second passage.
 14. The device of claim 13, wherein themembrane of each cell defines a second opening, through which theintracellular space of the cell is in fluid communication with the firstpassage through the first opening located between the sidewalls of acorresponding chamber.
 15. The device of claim 14, further comprising aplurality of punching elements, each positioned underneath the firstopening located between the sidewalls of a corresponding chamber formaking the second opening defined by the membrane of a correspondingcell.
 16. The device of claim 15, wherein each punching elementcomprises an electroporation device.
 17. The device of claim 13, whereinwhen a first medium is introduced into some portion of the first passageand the intracellular space of a cell that is in a chamber correspondingto that portion of the first passage is in fluid communication with thefirst passage, a corresponding sensor measures the response of the cellto the first medium.
 18. The device of claim 13, wherein when a secondmedium is introduced into a chamber and at least part of the membrane ofa corresponding cell in the chamber is in contact with the secondmedium, a corresponding sensor measures the response of the cell to thesecond medium.
 19. The device of claim 13, wherein when a first mediumis introduced into some portion of the first passage and a second mediumis introduced into a chamber corresponding to that respective portion ofthe first passage, respectively, the intracellular space of acorresponding cell in the chamber is in fluid communication with thefirst passage having the second medium, and at least part of themembrane of the corresponding cell is in contact with the second medium,a corresponding sensor measures the responses of the cell to the firstmedium and the second medium.
 20. The device of claim 13, wherein all ofthe plurality of sensors are sensors of substantially the same type. 21.The device of claim 13, wherein at least two of the plurality of sensorsare different from each other.
 22. The device of claim 13, wherein atleast one chamber is in fluid communication with a reservoir of a mediumthrough a corresponding second passage.
 23. The device of claim 13,wherein the first passage is in fluid communication with a reservoir ofa medium.
 24. The device of claim 13, wherein each seal element isformed and positioned to substantially encircle its corresponding firstopening.
 25. The device of claim 24, wherein each seal element comprisesan ohmic element.
 26. The device of claim 13, wherein all of theplurality of seal elements are seal elements of substantially the sametype or there are at least two different types of seal elements in theplurality of sensors.
 27. The device of claim 13, wherein the bodyportion of the second substrate further defines a plurality ofintersection portions where the first passage and the plurality of firstopenings are in fluid communication, respectively, and wherein eachintersection portion is at least partially formed as a cone shapedportion.